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Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The NetherlandsDepartment of Orthopaedics, University Medical Center Utrecht, Utrecht, The Netherlands
Orthopaedic Biomechanics, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The NetherlandsDepartment of Orthopaedics, University Medical Center Utrecht, Utrecht, The Netherlands
Department of Orthopaedics, University Medical Center Utrecht, Utrecht, The NetherlandsDepartment of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
In patients suffering from unilateral osteoarthritis in the knee, an osteotomy can provide symptomatic relief and postpone the need for replacement of the joint. Nevertheless, open-wedge osteotomies (OWO) around the knee joint face several challenges like postoperative pain and bone non-union. In this study, the aim was to design, fabricate, and evaluate a gap-filling implant for OWO using an osteoinductive and degradable biomaterial.
Methods
Design of porous wedge-shaped implants was based on computed tomography (CT) scans of cadaveric legs. Implants were 3D printed using a magnesium strontium phosphate-polycaprolactone (MgPSr-PCL) biomaterial ink. Standardized scaffolds with different inter-fibre spacing (IFS) were mechanically characterized and osteoinductive properties of the biomaterial were assessed in vitro. Finally, human-sized implants with different heights (5 mm, 10 mm, 15 mm) were designed and fabricated for ex vivo implantation during three OWO procedures in human cadaveric legs.
Results
Implants printed with an interior of IFS-1.0 resulted in scaffolds that maintained top and bottom porosity, while the interior of the implant exhibited significant mechanical stability. Bone marrow concentrate and culture expanded mesenchymal stromal cells attached to the MgPSr-PCL material and proliferated over 21 days in culture. The production of osteogenic markers alkaline phosphatase activity, calcium, and osteocalcin was promoted in all culture conditions, independent of osteogenic induction medium. Finally, three OWO procedures were planned and fabricated wedges were implanted ex vivo during the procedures. A small fraction of one side of the wedges was resected to assure fit into the proximal biplanar osteotomy gap. Pre-planned wedge heights were maintained after implantation as measured by micro-CT.
Conclusion
To conclude, personalized implants for implantation in open-wedge osteotomies were successfully designed and manufactured. The implant material supported osteogenesis of MSCs and BMC in vitro and full-size implants were successfully implemented into the surgical procedure, without compromising pre-planned wedge height.
Unicompartmental knee osteoarthritis (OA) is often associated with lower limb malalignment. Especially for younger patients (age < 65 years) with unicompartimental OA and a malalignment, a correctional osteotomy can be a surgical solution, aiming to unload the affected compartment
Opening- and closing-wedge high tibial osteotomy are comparable and early full weight bearing is safe with angular stable plate fixation: a meta-analysis.
Is opening-wedge high tibial osteotomy superior to closing-wedge high tibial osteotomy in treatment of unicompartmental osteoarthritis? A meta-analysis of randomized controlled trials.
, OWOs have gained popularity over CWOs in the tibia, mainly due to practical consideriations. When performing a CWO in the tibia, an osteotomy of the fibula is necessary and future conversion to a partial or total knee arthroplasty is complicated
. But not without importance, medial high tibial OWO remains associated with pain in the early postoperative stage and has a higher risk for delayed or non-union
. Postoperative pain is believed to be (at least in part) caused by bone marrow leakage from the osteotomy site, causing swelling, resulting in impaired early weight-bearing, ambulation, and rehabilitation
Modified Iliac Crest Reconstruction with Bone Cement for Reduction of Donor Site Pain and Morbidity after Open Wedge High Tibial Osteotomy: A Prospective Study.
. Filling the osteotomy gap with an allogeneic bone graft could be a viable solution as gap filler, and has demonstrated improved pain levels after the procedure compared to baseline in a case series of 103 patients
. Almost all of the patients (99%) were able to walk > 500 meters without any support three months after surgery. However, the use of allogeneic bone grafts is hampered by the limited availability of the grafts. Moreover, frozen allografts were shown to have a higher failure rate (defined as construct failure or non-union) compared to living autologous grafts
aid in bone union without donor site morbidity. Nevertheless, most bone substitute wedges are not designed to imitate the structure of trabecular bone with a dense cortical border. To improve bone union, postoperative pain, and eliminate the need for an allo- or autograft in OWO procedures, a firm, gap-filing 3D-printed scaffold with osteoconductive properties and mechanical stability provides a solution.
Among the bioactive ceramic materials that have been used for bone tissue engineering, magnesium strontium phosphate (MgPSr) has gained particular interest due to the good solubility of magnesium phosphate phases under physiological conditions, and the presence of Sr2+ ions have been demonstrated to promote osteogenic differentiation of mesenchymal stromal cells (MSCs)
. A previous study has investigated a ceramic-polymer composite of MgPSr and polycaprolactone (PCL), which is a versatile biomaterial ink that can be processed through extrusion-based 3D printing at room temperature. The biomaterial can be manufactured into different complex geometries to improve bone filling of a defect without compromising mechanical stability
. The porous nature of a printed osteotomy wedge scaffold allows bone marrow to populate the wedge upon implantation. Alternatively, pre-surgical seeding of the implant with a bioactive product accelerating osteogenesis, such as bone marrow concentrate (BMC), could further accelerate bone union.
This study aimed to design and manufacture a scaffold as gap filler in OWO around the knee joint. The mechanical stability of the wedge scaffold, as well as the in vitro osteoinductive properties of the material on MSCs and BMC were investigated. Additionally, preservation of the pre-designed implant structure and height were assessed upon implantation into human cadaveric legs.
Materials and Methods
Study outline
To completely fill the opening wedge gap after an OWO, 3D printed scaffolds were manufactured using patient computed tomography (CT) data and computer-aided design. The printed scaffold structures were mechanically characterized and in vitro potency of the MgPSr-PCL material was evaluated to induce osteogenesis when seeded with bone marrow-derived MSCs, as well as BMC. Finally, a proof-of-concept surgical implantation in a cadaver model was performed for implementation of the implants into the current osteotomy procedure.
Computer-aided design of osteotomy wedge
For initial design of a wedge scaffold for mechanical characterization and in vitro experiments, an anonymized CT scan and surgical planning for an 8 mm medial opening-wedge distal femur osteotomy was acquired from a clinical case (University Medical Center Utrecht) (Figure 1Ai). The computer-aided design (CAD) of the wedge scaffold was developed in SolidWorks software (Dassault Systèmes, Waltham, MA, USA), using the CT scan images. After assessment of the wedge scaffold, BioCAM™ software was used to define the wedge scaffold internal architecture and subsequently translate the design into a G-Code. The external wall of the osteotomy scaffold was kept closed with two outer layers, while for the internal region of the osteotomy scaffolds, three different inter-fibre spacings (IFS), 1.3 mm, 1.0 mm, and 0.7 mm (abbreviated as IFS-1.3, IFS-1.0, and IFS-0.7, respectively) were considered.
Material preparation and extrusion-based 3D-bioprinting
The biomaterial ink was prepared by combining in-house synthesized Mg2.33Sr0.67(PO4)2 powder and commercial medical grade poly(ε-caprolactone) (mPCL, Purasorb PC 12, Purac Biomaterials, Netherlands) in a weight ratio of 70:30 wt.% of MgPSr to PCL, according to a procedure previously described
. Designed scaffolds were fabricated by an extrusion-based 3D-printing system (3D Discovery, regenHU, Switzerland) using the MgPSr-PCL biomaterial ink. The ink was transferred to a 10 mL syringe (Nordson EFD, USA) and extruded though a 22G conical nozzle (inner diameter = 0.41 mm, Nordson EFD, USA) at a pressure of 0.9 bar and collected at collector speed of 6 mm/s.
Mechanical characterization of printed wedge scaffolds
Uniaxial compression tests were performed using a universal testing machine (Zwick Z010, Germany) equipped with a 1 kN load cell. Tests were performed on cylindrical samples (d = 6 mm, h = 12 mm, n = 5) for all three groups (IFS-1.3, IFS-1.0 and IFS-0.7, without closed outer edges), at a rate of 1 mm/min (at room temperature). From the engineered stress-strain curves, the elastic modulus (defined as the slope of the linear region at the interval 0.02 - 0.05 mm/mm strain), the yield stress (defined as the point where nonlinear deformation begins), and toughness (defined as the absorbed energy by the scaffolds up to yield stress) were determined.
In vitro accelerated degradation of printed wedge scaffolds
The degradation of the materials was studied under controlled conditions which accelerated biomaterial degradation in vitro
. Wedge scaffolds were incubated in a 0.4 mg/ml lipase solution (from Pseudomonas cepacia, Sigma-Aldrich) and 1 mg/ml sodium azide (Sigma-Aldrich) at 37ºC for 15 days. At each time point (1, 5, 10, and 15 days), the enzymatic solution was refreshed, and samples were monitored for weight loss, quantified as follows:
(1)
In vitro osteogenesis of scaffolds
Donors and cell isolation
Human bone marrow-derived mesenchymal stromal cells (BM-MSCs) were derived from healthy donor bone marrow aspirates (n = 3, age range 2 – 12) as approved by the Dutch central Committee on Research Involving Human Subjects (CCMO, Bio-banking bone marrow for MSC expansion, NL41015.041.12). The parent or legal guardian of the donor signed the informed consent approved by the CCMO. In brief, the mononuclear fraction was separated using a density gradient (Lympoprep, Axis Shield). MSCs were isolated by plastic adherence and expanded for three passages in Minimum Essential Media (αMEM, Macopharma) with 5% platelet lysate and 3.3 IU/mL heparin and cryopreserved. Subsequently, MSCs were expanded for two additional passages in MSC expansion medium (αMEM [Gibco], 10% (v/v) fetal bovine serum [FBS; Biowest], 1% penicillin/streptomycin [pen/strep; 100 U/mL, 100 µg/mL], 200 µM l-ascorbic acid 2-phosphate [ASAP; Sigma-Aldrich], and 1 ng/mL basic fibroblast growth factor [bFGF; PeproTech]). BMC was obtained from donors undergoing an OWO or total knee arthroplasty surgery (n = 2, age range 39 - 49) after their informed consent (protocol approved by the local medical ethical committee). Bone marrow was concentrated to one tenth of its original volume using Ficoll paque (GE Healthcare) density separation.
In vitro culture of scaffolds
Standardized 5 mm diameter cylindrical scaffolds (ISF-1.0) were printed as described before
and sterilized by washing in 70% ethanol and Milli-Q, followed by exposure to ultraviolet light for 1 hour. Scaffolds were cut in half with a sterile scalpel and seeded with either 15,000 MSCs / scaffold in fibrin gel (25 µL fibrinogen [1:15 in PBS] crosslinked with 25 µL thrombin (1:50 in PBS); Tisseel, Baxter) or 25 µL BMC (crosslinked with 16.6 µL thrombin and 16.6 µL CaCl2 [500 mM in 0.9% NaCl]). Cell-seeded scaffolds were pre-cultured in MSC expansion medium for two days, followed by 21 days of osteogenic induction with osteogenic differentiation medium (αMEM supplemented with 10% FBS, 1% pen/strep, 200 µM ASAP, 10 mM β-glycerophosphate [Sigma-Aldrich], and 10 nM dexamethasone [Sigma-Aldrich]). Control cell-seeded scaffolds were treated with MSC expansion medium without bFGF.
Alkaline phosphatase, calcium, and DNA quantification
Osteogenic differentiation of the cells was measured by the activity of the early osteogenic marker alkaline phosphatase (ALP) after 5, 7, and 11 days and by quantification of calcium produced after 21 days. To determine activity of ALP, cells were lysed in Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) by three freeze-thaw cycles. ALP activity was measured using the conversion of p-nitrophenyl phosphate liquid substrate (pNPP, Sigma-Aldrich). Absorbance was measured every minute for 30 minutes at 405 nm and corrected for absorbance at 655 nm. Calf intestinal ALP (Sigma-Aldrich) was used as a standard. Calcium concentration in the samples was quantified after 21 days using a colorimetric calcium assay kit (Abcam) according to the manufacturer's instructions. ALP activity and calcium levels were corrected for DNA. DNA content was determined using the Quant-iT PicoGreen dsDNA assay (Invitrogen) according to the manufacturer's instructions.
Osteocalcin immunocytochemistry
To visualise the osteogenic marker osteocalcin, scaffolds were fixed in formalin for 30 minutes for the osteocalcin immunocytochemistry after 21 days of differentiation. Samples were permeabilized with 0.2% (v/v) Triton X-100 in phosphate-buffered saline (PBS), followed by blocking with 5% (v/v) bovine serum albumin (BSA) in PBS. Next, samples were incubated overnight at 4ºC with 10 µg/mL mouse-anti-human primary antibody against osteocalcin (clone OCG4; Enzo Life Sciences). Samples were then incubated with 10 µg/mL goat-anti-mouse antibody conjugated to Alex Fluor 488 (Invitrogen) for one hour at room temperature. All samples were also stained for F-actin (1:200; phalloidin-TRITC; Sigma-Aldrich) and 4′,6-diamidino-2-phenylindole (100 ng/mL; DAPI; Sigma-Aldrich). Images were acquired with a Leica SP8X Laser Scanning Confocal Microscope and Leica LASX acquisition software.
Ex vivo surgical implantation of the printed wedges
Three fresh-frozen human cadaveric legs were obtained (all left legs, one male and two female) in accordance with the guidelines of the local medical ethical committee. CT-scans were obtained of the three included legs (Philips Healthcare, Best, The Netherlands; 100 kV and 130mAs), with 0.8 mm slice thickness. Single plane OWOs were pre-operatively planned in 3-Matic (Materialise, Leuven, Belgium), with for each leg a specific wedge height (5, 10, and 15 mm). This resulted in post-surgical 3D-models of the cadavers with left open osteotomy gaps, which functioned as surrogate for the 3D printing of the wedges. A proximal biplanar medial high tibial OWO was performed following a standard surgery protocol. During this procedure, the osteotomy gap was kept open using a lamina spreader and the 3D-printed scaffold wedge was inserted into the gap. The tips of the wedge scaffolds were resected to fit the biplanar osteotomy gap, without altering the outside rim of the wedge. The osteotomies were then fixated with angular stable plates (Activmotion, Newclip Technics, Haute-Goulaine, France). The same plates are used for patients in the clinic and have a smaller footprint compared to other commercially used plates. Following implantation of the scaffold wedges, additional CT scans were obtained of the operated cadaver legs, subsequently the wedges were explanted for further analysis.
Micro-computed tomography
The pre- and post-surgical wedge scaffolds underwent micro-CT (Quantum FX-Perkin Elmer, USA) for height analyses. Scan parameters were 90 kV tube voltage, 180 µA tube current, 60 or 73 mm resolution, and 2 min scan time. Scaffold heights pre- and post-implantation were quantified using computer vision software Fiji (software version 2.1.0/1.53c, National Institutes of Health, Bethesda, USA). Scaffold height was measured at the highest point. The micro-CT images of a single scaffold pre- and post-implantation were superimposed to ensure the scaffolds were measured at the same location at both timepoints.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 8.3 (GraphPad Software, Inc., La Jolla, CA, USA). All data were presented as mean ± standard deviation (SD). To test for differences in mechanical evaluations and calcium content, a one-way analysis of variance (ANOVA) with Tukey's post hoc test was used. To test for differences in DNA and ALP quantifications, a two-way ANOVA with Tukey's post hoc was used. For scaffold wedge height, a two-tailored t-test was used. Normality was confirmed with a Shapiro-Wilk test (p>0.05). P values below 0.05 were considered significant.
Results
Personalized implant design and fabrication
Wedge implants were designed for both open-wedge lateral distal femur (for in vitro analyses; Figure 1A) and medial tibial osteotomies (for implantation in cadavers; Figure 1B) from CT scan 3D reconstructions (Panels i). Wedges had closed outer edges, aimed at limiting leakage from the osteotomy site into the soft tissues surrounding the bone, while the interior was porous (Panels ii and iii).
Figure 1Surgical planning and extrusion-based printing. Wedge design for open-wedge osteotomies in (A) distal femur and (B) proximal tibia. (Panels i) Surgical planning of open-wedge osteotomies derived from computed tomography (CT) scans. (Panels ii) Top views of printing paths of computer-aided designs (CAD) of personalized wedge implants. (Panels iii) The finalized personalized wedges in magnesium strontium phosphate-polycaprolactone (MgPSr-PCL) biomaterial. Scale bar = 10 mm.
Incorporation of the thermoplastic PCL into the ceramic MgPSr phase improved handling of the implants, overcoming downsides of brittle ceramic materials
. The stress-strain curves of the standardized printed discs with different IFS showed comparable profiles (Figure 2A). The decrease in IFS resulted in an increase in mechanical stability, elastic modulus increased significantly from 105.3 ± 10.26 MPa (IFS-1.3) to 151.5 ± 12.61 MPa (IFS-0.7) (Figure 2C). Yield stress, defined as the point of maximum elastic deformation, increased from 4.2 ± 1.27 MPa (IFS-1.3) to 6.0 ± 1.90 MPa and 11.4 ± 1.86 MPa for IFS-1.0 and IFS-0.7, respectively (Figure 2D). In line, strain energy increased from 0.077 ± 0.0208 J for IFS-1.3 to 0.196 ± 0.0957 J for IFS-0.7 (Figure 2E). While a disc with an IFS of 0.7 mm presented the highest elastic modulus, printing of a complete wedge scaffold with this IFS resulted in a construct that was not completely porous from top to bottom, which is essential to flow of bone marrow through the scaffold in vivo (Figure 2B). Scaffolds with a planned IFS of 1.0 mm resulted in completely porous wedges and only a slight difference in elastic modulus and strain energy compared to scaffolds with a planned IFS of 0.7 mm, which did not reach statistical significance. While the degradation rate of IFS-1.3 samples (as evaluated under accelerated degradation conditions) was 30% faster compared to IFS-1.0 samples (Figure 2F), the combination of tested characteristics led to the selection of IFS-1.0 for further analyses as the best compromise between open porosity, mechanical stability, and degradation properties.
Figure 2Evaluation of mechanical properties of the printed magnesium strontium phosphate-polycaprolactone (MgPSr-PCL) wedges. A) Longitudinal compression profile of 3D printed MgPSr-PCL wedge scaffolds for inter-fibre spacing (IFS) -1.3, IFS-1.0, and IFS-0.7. B) Corresponding photographs showed the different scaffolds after the printing. Open pores in wedges IFS-1.3 and IFS-1.0 can be appreciated, while IFS-0.7 wedges were not porous. C) Elastic modulus, D) Yield stress, and E) strain energy from compressive loading profile for IFS-1.3, IFS-1.0, and IFS-0.7. F) Weight loss of wedge scaffolds during accelerated in vitro degradation in enzymatic solution over 15 days. *p<0.05
In vitro osteogenic properties of the scaffold material
Human BM-MSCs embedded in fibrin (MSC-fibrin) were seeded in the biomaterial scaffolds to evaluate osteogenic potential. Additionally, to simulate the in vivo situation, a second group of scaffolds was seeded with BMC. The culture-expanded human MSCs attached to the MgPSr-PCL material and proliferated over time. Cells in BMC also proliferated on the scaffolds (Figure 4A and Supplemental Figure 1). Activity of the early osteogenic marker ALP was similar in MSC-fibrin and BMC groups when scaffolds were cultured in control medium, yet were increased in MSC-fibrin when cultured in osteogenic medium (Figure 4B). BMC performed similar to MSC-fibrin in terms of calcium production at 21 days of culture (Figure 4C). Of note, both experimental groups had a higher ALP activity and increased calcium production compared to MSCs that were cultured in monolayers, indicating osteoconductive effects of the scaffold material and 3D environment. Production of osteocalcin, an exclusive marker for osteoblasts, was observed in cultures under all conditions irrespective of culture medium used (Figure 3D).
Figure 3In vitro osteogenic performance of the magnesium strontium phosphate-polycaprolactone (MgPSr-PCL) biomaterial. A) Quantification of DNA in the MgPSr-PCL scaffolds at 5, 7, 11, and 21 days in culture with control (left panel) and osteogenic medium (right panel). B) Early osteogenic marker alkaline phosphatase (ALP) activity relative to the amount of DNA at 5, 7, and 11 days. C) Calcium content of the cultured constructs at day 21 in control medium (top panel) and osteogenic medium (bottom panel). D) Immunocytochemical osteocalcin staining on 21-day cultured standardized cylindrical MgPSr-PCL scaffolds (ISF-1.0) using: (i) culture expanded mesenchymal stromal cells (MSC) in control medium, (ii) MSC in osteogenic medium, (iii) bone marrow concentrate (BMC) in control medium, and (iv) BMC in osteogenic medium. Nuclei are shown in blue (DAPI), osteocalcin expression in green, and F-actin in red. Dashed lines indicate the location of three-dimensional scaffold material. Scale bar = 100 µm. *p<0.05
Three fresh-frozen human cadaveric legs underwent CT scanning in order to plan three OWOs with different heights; 5, 10, and 15 mm (Figure 4A, 4B, 4C). 3D models of the tibias were used to plan single plane osteotomy gap (Panels i) and design the wedge scaffolds (Panels ii). Wedges were implanted during a standard proximal biplanar OWO procedure and fixated with an angular stable plate (Panels iii). Post-surgical X rays (Panels iv) and CT scans (Panels v) illustrate the scaffold positioning and fit. Micro-CT analyses of the wedges pre- and post-implantation indicated good analogy of the scaffolds (Figure 5A, pre-operative in red, post-operative in grey). Due to the biplanar approach of the osteotomy procedure
, the scaffolds were adjusted at one side using an automatic saw, not altering the rest of the scaffold shape and outer rim (Figure 5B, arrows indicating trimmed edge). Quantification of wedge heights from micro-CT images revealed that the wedge heights were not affected by the applied surgical procedure, during which they bear loading for a brief moment when the laminar spreader is removed to allow sufficient space for the plate to be fixated (Figure 5C).
Figure 4Surgical implantation of personalized scaffold wedges. Planned osteotomy heights of A) 5 mm, B) 10 mm, and C) 15 mm from computed tomography (CT) scans of human cadaveric legs (Panels i). (Panels ii) 3D printed wedge scaffolds in magnesium strontium phosphate-polycaprolactone (MgPSr-PCL). (Panels iii) Scaffolds implanted in the cadaveric legs. (Panels iv) X-ray of the legs after implantation. (Panels v) 3D reconstruction from CT scans after implantation of the wedge scaffolds.
Figure 5Micro-computed tomography (CT) analysis of printed wedge scaffolds. A) 3D reconstructions of the wedges from micro-CT images of the printed wedges pre- and post-implantation. Pre-implanted scaffolds in red, post-implanted scaffolds in grey. B) Micro-CT reconstruction of the 5 mm wedge. Note the small portion that was adjusted during the procedure (indicated by the white arrows). C) Quantification of scaffold wedge height before and after implantation into the cadaveric legs revealed no loss of scaffold height.
This study demonstrated a proof of concept to manufacture implants for bone gap filling in femoral or tibial OWOs, by 3D printing a biodegradable and osteoinductive scaffold material. The printed material promoted osteogenesis of MSCs and BMC in vitro and scaffolds were implanted ex vivo without compromising pre-operatively planned wedge height. The aim was to design implants that fitted the planned osteotomy gap and height, fabricate these, and evaluate their implementation in the established surgical procedure.
Post-surgical pain poses a challenge in OWO care. One of the hypotheses for the cause of pain is bleeding from the osteotomy site. It has been shown that fitting an allogeneic graft, which closes the gap completely, enables early postoperative weight-bearing and improve clinical outcomes after three months
. A variety of scaffold materials and growth factors is being investigated to accelerate osteogenesis in OWO. Most studies investigating a scaffold material make use of wedges composed of hydroxyapatite or beta-tricalcium phosphate
Comparative outcomes of open-wedge high tibial osteotomy with platelet-rich plasma alone or in combination with mesenchymal stem cell treatment: A prospective study.
Recombinant Human BMP6 Applied Within Autologous Blood Coagulum Accelerates Bone Healing: Randomized Controlled Trial in High Tibial Osteotomy Patients.
While a great deal of effort is put into accelerating bone healing, the current study investigates a combined approach of osteoinduction and filling of the osteotomy gap. The presented methods offer the possibility of personalizing an osteoinductive wedge implant and incorporating this into the existing 3D workflow. Yet, the manufactured implants were designed to match opening-wedge height and the anatomy of the bone. Adjustment of the scaffold edge enabled proper fit into the osteotomy plane, while still filling the gap. Precisely sealing of the whole gap was proven to be challenging in the current setup. The approach used in the current study led to a relatively symmetrical wedge, as opposed to a trapezoid-shaped wedge. To move towards clinical implementation and complete sealing of an osteotomy gap, a more precise, biplanar surgical planning should be performed. Most likely, this would also require using 3D printed patient-specific instruments (PSI), with pre-operatively determined saw cuts resulting in a predefined gap morphology, for which a fitting implant can subsequently be fabricated. In some cases of malalignment correction, PSI are preferred by orthopaedic surgeons, in the form of saw and drill guides per-operatively. Further research can offer the possibility of adding a personalized wedge implant into this workflow. With predetermined bone cuts and gap morphology, our wedge scaffold can be 3D printed pre-operatively to fit. However, this initial study focussed primarily on the feasibility of implanting a 3D printed MgPSr-PCL wedge scaffold, without compromising the pre-operatively planned wedge height.
While most of the load on the osteotomy gap is absorbed by the angular stable plate and screws, the implant should remain stable during the brief period before the plate is fixed on the bone. The elastic modulus of the implants demonstrated similarities to human trabecular bone
. Scaffolds with a planned fibre spacing of 1.0 mm maintained open pores, through which bone marrow would be allowed to flow and osteogenesis might be accelerated. While implant height was maintained post-implantation in the cadaveric legs, future in vivo and clinical studies are necessary to confirm maintenance of pre-planned wedge height during a longer period of implantation, as well as speed of bone union.
The full-size implants degraded over time in an accelerated in vitro setup using an enzymatic solution. Prior research in an equine model has shown that degradation of the MgPSr-PCL (pore size 1.0 mm) in the enzymatic solution for ten days corresponded to degradation over six months during in vivo implantation
. Increasing inter-fibre spacing accelerated degradation by 30% in mass loss, indicating that in vivo mass loss might also be accelerated. However, the exact degradation and speed of bone formation of the osteotomy-specific MgPSr-PCL implants in this specific anatomical location should be evaluated in a large animal model.
The printed MgPSr-PCL material facilitated osteogenesis in vitro, confirming previous findings
. For large defects, pre-seeding of the scaffold with a regenerative compound, like MSCs, might be beneficial to accelerate bone healing. Here, seeding of the material with both culture-expanded MSCs and BMC resulted in production of osteoblast-specific markers, indicating that infiltrated bone marrow in the scaffold material after implantation may be sufficient by itself to stimulate osteogenesis. This is most likely induced by its growth factor-rich nature and the presence of progenitor cells
, this study is the first to report on a personalized biodegradable implant for OWO.
The current study was mainly limited by the imbalance between preoperative osteotomy planning (single plane) and the intra-operative surgical biplanar osteotomy, leading to a slight mismatch between gap and fabricated implant. By implementing 3D printed PSI in the workflow, an optimal fit of the designed implant can be achieved. Because the purpose of this study was to design and manufacture a gap-filling wedge implant in an osteoinductive material and to evaluate this in an ex vivo model, achieving a perfect fit was beyond the scope of this investigation.
Conclusions
To conclude, a gap-filling implant for open-wedge osteotomies was designed and manufactured. This implant was 3D manufactured in an osteoinductive and biodegradable material that supported cell attachment, growth, and production of early and late osteogenic markers in vitro. Finally, an ex vivo proof-of-concept of the surgical procedure was successfully performed, implementing the designed wedge scaffolds into the standard osteotomy procedure, while maintaining implant integrity and pre-planned wedge height.
Author's Contributions
LV, NvE, RC, and JM conceived the study. All authors were involved in design of the study. MR, NG, and MdR carried out the laboratory experiments. NG and MC prepared the biomaterial ink. MR, HN, NvE, and RC were involved in the cadaver study. MR, HN, and NG drafted the manuscript. All authors revised the manuscript critically and have given final approval of the version to be published.
Supplemental Figure 1. DNA quantification. Quantification of DNA in scaffolds seeded with bone marrow-derived mesenchymal stromal cells (MSC) and bone marrow concentrate (BMC) compared to MSCs cultured in monolayers as controls.
Declaration of Competing Interest
Nienke van Egmond reports a relationship with Newclip Technics that includes: consulting or advisory.
Lucienne Vonk reports a relationship with CO.DON AG that includes: employment.
Lucienne Vonk reports a relationship with International Cartilage Regeneration & Joint Preservation Society that includes: non-financial support.
Senior Associate Editor for The Journal of Cartilage and Joint Preservation - L.V.
Editor for The Journal of Cartilage and Joint Preservation - R.C.
Editor for The Journal of Cartilage and Joint Preservation - J.M.
Funding
This research is supported by the partners of Regenerative Medicine Crossing Borders (RegMed XB), financed by the Dutch Ministry of Economic Affairs by means of the PPP Allowance made available by the Top Sector Life Sciences & Health to stimulate public-private partnership, and ReumaNederland (LLP-12, LLP22, and 19-1-207 MINIJOINT). JM and MC acknowledge financial support from the Gravitation Program “Materials Driven Regeneration”, funded by the Netherlands Organization for Scientific Research (024.003.013). MC also acknowledges funding from Reprint project (OCENW.XS5. 161) by Netherlands Organization Scientific Research.
Acknowledgements
The authors would like to thank Arno Mooring (iMoveMedical, Nieuwegein, The Netherlands) for providing surgical tools and the 3D Lab of the University Medical Center for their assistance in planning of the osteotomies. The authors would also like to thank Nils van Veen for assisting with the CT scans and Marco Rondhuis for his kind assistance at the Anatomy Department. The authors also thank Dr. Elke Vorndran and Prof. Uwe Gbureck from the Department for Functional Materials in Medicine and Dentistry, University of Würzburg, for kindly synthetizing and providing the ceramic biomaterial used in this work.
Informed Patient Consent
This research did not include any human subjects and therefore no written consent was necessary to be obtained.
Opening- and closing-wedge high tibial osteotomy are comparable and early full weight bearing is safe with angular stable plate fixation: a meta-analysis.
Is opening-wedge high tibial osteotomy superior to closing-wedge high tibial osteotomy in treatment of unicompartmental osteoarthritis? A meta-analysis of randomized controlled trials.
Modified Iliac Crest Reconstruction with Bone Cement for Reduction of Donor Site Pain and Morbidity after Open Wedge High Tibial Osteotomy: A Prospective Study.
Comparative outcomes of open-wedge high tibial osteotomy with platelet-rich plasma alone or in combination with mesenchymal stem cell treatment: A prospective study.
Recombinant Human BMP6 Applied Within Autologous Blood Coagulum Accelerates Bone Healing: Randomized Controlled Trial in High Tibial Osteotomy Patients.
Surgical planning and extrusion-based printing. Wedge design for open-wedge osteotomies in (A) distal femur and (B) proximal tibia. (Panels i) Surgical planning of open-wedge osteotomies derived from computed tomography (CT) scans. (Panels ii) Top views of printing paths of computer-aided designs (CAD) of personalized wedge implants. (Panels iii) The finalized personalized wedges in magnesium strontium phosphate-polycaprolactone (MgPSr-PCL) biomaterial. Scale bar = 10 mm.
Evaluation of mechanical properties of the printed magnesium strontium phosphate-polycaprolactone (MgPSr-PCL) wedges. A) Longitudinal compression profile of 3D printed MgPSr-PCL wedge scaffolds for inter-fibre spacing (IFS) -1.3, IFS-1.0, and IFS-0.7. B) Corresponding photographs showed the different scaffolds after the printing. Open pores in wedges IFS-1.3 and IFS-1.0 can be appreciated, while IFS-0.7 wedges were not porous. C) Elastic modulus, D) Yield stress, and E) strain energy from compressive loading profile for IFS-1.3, IFS-1.0, and IFS-0.7. F) Weight loss of wedge scaffolds during accelerated in vitro degradation in enzymatic solution over 15 days. *p<0.05
In vitro osteogenic performance of the magnesium strontium phosphate-polycaprolactone (MgPSr-PCL) biomaterial. A) Quantification of DNA in the MgPSr-PCL scaffolds at 5, 7, 11, and 21 days in culture with control (left panel) and osteogenic medium (right panel). B) Early osteogenic marker alkaline phosphatase (ALP) activity relative to the amount of DNA at 5, 7, and 11 days. C) Calcium content of the cultured constructs at day 21 in control medium (top panel) and osteogenic medium (bottom panel). D) Immunocytochemical osteocalcin staining on 21-day cultured standardized cylindrical MgPSr-PCL scaffolds (ISF-1.0) using: (i) culture expanded mesenchymal stromal cells (MSC) in control medium, (ii) MSC in osteogenic medium, (iii) bone marrow concentrate (BMC) in control medium, and (iv) BMC in osteogenic medium. Nuclei are shown in blue (DAPI), osteocalcin expression in green, and F-actin in red. Dashed lines indicate the location of three-dimensional scaffold material. Scale bar = 100 µm. *p<0.05
Surgical implantation of personalized scaffold wedges. Planned osteotomy heights of A) 5 mm, B) 10 mm, and C) 15 mm from computed tomography (CT) scans of human cadaveric legs (Panels i). (Panels ii) 3D printed wedge scaffolds in magnesium strontium phosphate-polycaprolactone (MgPSr-PCL). (Panels iii) Scaffolds implanted in the cadaveric legs. (Panels iv) X-ray of the legs after implantation. (Panels v) 3D reconstruction from CT scans after implantation of the wedge scaffolds.
Micro-computed tomography (CT) analysis of printed wedge scaffolds. A) 3D reconstructions of the wedges from micro-CT images of the printed wedges pre- and post-implantation. Pre-implanted scaffolds in red, post-implanted scaffolds in grey. B) Micro-CT reconstruction of the 5 mm wedge. Note the small portion that was adjusted during the procedure (indicated by the white arrows). C) Quantification of scaffold wedge height before and after implantation into the cadaveric legs revealed no loss of scaffold height.
In patients suffering from unilateral osteoarthritis in the knee, an osteotomy can provide symptomatic relief and postpone the need for replacement of the joint. Nevertheless, open-wedge osteotomies (OWO) around the knee joint face several challenges like postoperative pain and bone non-union. In this study, the aim was to design, fabricate, and evaluate a gap-filling implant for OWO using an osteoinductive and degradable biomaterial.
Methods
Design of porous wedge-shaped implants was based on computed tomography (CT) scans of cadaveric legs. Implants were 3D printed using a magnesium strontium phosphate-polycaprolactone (MgPSr-PCL) biomaterial ink. Standardized scaffolds with different inter-fibre spacing (IFS) were mechanically characterized and osteoinductive properties of the biomaterial were assessed in vitro. Finally, human-sized implants with different heights (5 mm, 10 mm, 15 mm) were designed and fabricated for ex vivo implantation during three OWO procedures in human cadaveric legs.
Results
Implants printed with an interior of IFS-1.0 resulted in scaffolds that maintained top and bottom porosity, while the interior of the implant exhibited significant mechanical stability. Bone marrow concentrate and culture expanded mesenchymal stromal cells attached to the MgPSr-PCL material and proliferated over 21 days in culture. The production of osteogenic markers alkaline phosphatase activity, calcium, and osteocalcin was promoted in all culture conditions, independent of osteogenic induction medium. Finally, three OWO procedures were planned and fabricated wedges were implanted ex vivo during the procedures. A small fraction of one side of the wedges was resected to assure fit into the proximal biplanar osteotomy gap. Pre-planned wedge heights were maintained after implantation as measured by micro-CT.
Conclusion
To conclude, personalized implants for implantation in open-wedge osteotomies were successfully designed and manufactured. The implant material supported osteogenesis of MSCs and BMC in vitro and full-size implants were successfully implemented into the surgical procedure, without compromising pre-planned wedge height.
While open-wedge osteotomies (OWO) and closing-wedge osteotomies (CWO) have shown comparible clinical outcome4x4van Haeringen, MH, Kuijer, PPFM, Daams, JG et al. Opening- and closing-wedge high tibial osteotomy are comparable and early full weight bearing is safe with angular stable plate fixation: a meta-analysis. Knee Surgery, Sports Traumatology, Arthroscopy. 2022;
https://doi.org/10.1007/s00167-022-07229-3 (Published online) Crossref | Scopus (0) | Google ScholarSee all References,5x5Wang, Z, Zeng, Y, She, W, Luo, X, and Cai, L. Is opening-wedge high tibial osteotomy superior to closing-wedge high tibial osteotomy in treatment of unicompartmental osteoarthritis? A meta-analysis of randomized controlled trials. International Journal of Surgery. 2018;
60: 153–163https://doi.org/10.1016/j.ijsu.2018.10.045 Crossref | PubMed | Scopus (25) | Google ScholarSee all References, OWOs have gained popularity over CWOs in the tibia, mainly due to practical consideriations. When performing a CWO in the tibia, an osteotomy of the fibula is necessary and future conversion to a partial or total knee arthroplasty is complicated6x6Lee, DC and Byun, SJ. High Tibial Osteotomy. Knee Surg Relat Res. 2012;
24: 61–69https://doi.org/10.5792/ksrr.2012.24.2.61 Crossref | PubMed | Scopus (138) | Google ScholarSee all References. But not without importance, medial high tibial OWO remains associated with pain in the early postoperative stage and has a higher risk for delayed or non-union7x7Han, JH, Kim, HJ, Song, JG et al. Is Bone Grafting Necessary in Opening Wedge High Tibial Osteotomy? A Meta-Analysis of Radiological Outcomes. Knee Surg Relat Res. 2015;
27: 207–220https://doi.org/10.5792/ksrr.2015.27.4.207 Crossref | Scopus (33) | Google ScholarSee all References,8x8van Genechten, W, van den Bempt, M, van Tilborg, W et al. Structural allograft impaction enables fast rehabilitation in opening-wedge high tibial osteotomy: a consecutive case series with one year follow-up. Knee Surgery, Sports Traumatology, Arthroscopy. 2020;
28: 3747–3757https://doi.org/10.1007/s00167-019-05765-z Crossref | Scopus (8) | Google ScholarSee all References. Postoperative pain is believed to be (at least in part) caused by bone marrow leakage from the osteotomy site, causing swelling, resulting in impaired early weight-bearing, ambulation, and rehabilitation9x9Cao, ZW, Mai, XJ, Wang, J, Feng, EH, and Huang, YM. Uni-compartmental knee arthroplasty versus high tibial osteotomy for knee osteoarthritis: a systematic review and meta-analysis. Journal of Arthroplasty. 2018;
33: 952–959https://doi.org/10.1016/j.arth.2017.10.025 Abstract | Full Text | Full Text PDF | PubMed | Scopus (89) | Google ScholarSee all References.
In some cases, the opened osteotomy wedge is filled with an autologous bone graft from the iliac crest10x10Chae, DJ, Shetty, GM, Lee, DB, Choi, HW, Han, SB, and Nha, KW. Tibial slope and patellar height after opening wedge high tibia osteotomy using autologous tricortical iliac bone graft. Knee. 2008;
15: 128–133https://doi.org/10.1016/j.knee.2007.11.001 Abstract | Full Text | Full Text PDF | PubMed | Scopus (73) | Google ScholarSee all References. However, this procedure is aimed at accelerating union rather than closing the gap and it is associated with donor site morbidity11x11Lee, JS, Park, YJ, Wang, L, Chang, YS, Shetty, GM, and Nha, KW. Modified Iliac Crest Reconstruction with Bone Cement for Reduction of Donor Site Pain and Morbidity after Open Wedge High Tibial Osteotomy: A Prospective Study. Knee Surg Relat Res. 2016;
28: 277–282https://doi.org/10.5792/ksrr.15.046 Crossref | PubMed | Scopus (6) | Google ScholarSee all References. Filling the osteotomy gap with an allogeneic bone graft could be a viable solution as gap filler, and has demonstrated improved pain levels after the procedure compared to baseline in a case series of 103 patients8x8van Genechten, W, van den Bempt, M, van Tilborg, W et al. Structural allograft impaction enables fast rehabilitation in opening-wedge high tibial osteotomy: a consecutive case series with one year follow-up. Knee Surgery, Sports Traumatology, Arthroscopy. 2020;
28: 3747–3757https://doi.org/10.1007/s00167-019-05765-z Crossref | Scopus (8) | Google ScholarSee all References. Almost all of the patients (99%) were able to walk > 500 meters without any support three months after surgery. However, the use of allogeneic bone grafts is hampered by the limited availability of the grafts. Moreover, frozen allografts were shown to have a higher failure rate (defined as construct failure or non-union) compared to living autologous grafts12x12Kuremsky, MA, Schaller, TM, Hall, CC, Roehr, BA, and Masonis, JL. Comparison of autograft vs allograft in opening-wedge high tibial osteotomy. Journal of Arthroplasty. 2010;
25: 951–957https://doi.org/10.1016/j.arth.2009.07.026 Abstract | Full Text | Full Text PDF | PubMed | Scopus (52) | Google ScholarSee all References. Alternatively, synthetic bone substitutes made of hydroxyapatite and/or beta-tricalcium phosphate7x7Han, JH, Kim, HJ, Song, JG et al. Is Bone Grafting Necessary in Opening Wedge High Tibial Osteotomy? A Meta-Analysis of Radiological Outcomes. Knee Surg Relat Res. 2015;
27: 207–220https://doi.org/10.5792/ksrr.2015.27.4.207 Crossref | Scopus (33) | Google ScholarSee all References,13x13Lode, A, Meissner, K, Luo, Y et al. Fabrication of porous scaffolds by three-dimensional plotting of a pasty calcium phosphate bone cement under mild conditions. J Tissue Eng Regen Med. 2014;
8: 682–693https://doi.org/10.1002/term.1563 Crossref | Scopus (98) | Google ScholarSee all References,14x14Hooper, NM, Schouten, R, and Hooper, GJ. The Outcome of Bone Substitute Wedges in Medial Opening High Tibial Osteotomy. Open Orthop J. 2013;
7: 373–377https://doi.org/10.2174/1874325001307010373 Crossref | Google ScholarSee all References,15x15Castilho, M, Moseke, C, Ewald, A et al. Direct 3D powder printing of biphasic calcium phosphate scaffolds for substitution of complex bone defects. Biofabrication. 2014;
6https://doi.org/10.1088/1758-5082/6/1/015006 Crossref | Scopus (177) | Google ScholarSee all References aid in bone union without donor site morbidity. Nevertheless, most bone substitute wedges are not designed to imitate the structure of trabecular bone with a dense cortical border. To improve bone union, postoperative pain, and eliminate the need for an allo- or autograft in OWO procedures, a firm, gap-filing 3D-printed scaffold with osteoconductive properties and mechanical stability provides a solution.
Among the bioactive ceramic materials that have been used for bone tissue engineering, magnesium strontium phosphate (MgPSr) has gained particular interest due to the good solubility of magnesium phosphate phases under physiological conditions, and the presence of Sr2+ ions have been demonstrated to promote osteogenic differentiation of mesenchymal stromal cells (MSCs)16x16Yang, X, Xie, B, Wang, L, Qin, Y, Henneman, ZJ, and Nancollas, GH. Influence of magnesium ions and amino acids on the nucleation and growth of hydroxyapatite. CrystEngComm. 2011;
13: 1153–1158https://doi.org/10.1039/c0ce00470g Crossref | Scopus (76) | Google ScholarSee all References,17x17Yang, F, Yang, D, Tu, J, Zheng, Q, Cai, L, and Wang, L. Strontium enhances osteogenic differentiation of mesenchymal stem cells and in vivo bone formation by activating Wnt/catenin signaling. Stem Cells. 2011;
29: 981–991https://doi.org/10.1002/stem.646 Crossref | PubMed | Scopus (371) | Google ScholarSee all References,18x18Reitmaier, S, Kovtun, A, Schuelke, J et al. Strontium(II) and mechanical loading additively augment bone formation in calcium phosphate scaffolds. Journal of Orthopaedic Research. 2018;
36: 106–117https://doi.org/10.1002/jor.23623 Crossref | Scopus (30) | Google ScholarSee all References,19x19Lode, A, Heiss, C, Knapp, G et al. Strontium-modified premixed calcium phosphate cements for the therapy of osteoporotic bone defects. Acta Biomater. 2018;
65: 475–485https://doi.org/10.1016/j.actbio.2017.10.036 Crossref | Scopus (67) | Google ScholarSee all References,20x20Kim, JA, Lim, J, Naren, R, suk, Yun H, and Park, EK. Effect of the biodegradation rate controlled by pore structures in magnesium phosphate ceramic scaffolds on bone tissue regeneration in vivo. Acta Biomater. 2016;
44: 155–167https://doi.org/10.1016/j.actbio.2016.08.039 Crossref | Scopus (95) | Google ScholarSee all References,21x21Jia, J, Zhou, H, Wei, J et al. Development of magnesium calcium phosphate biocement for bone regeneration. J R Soc Interface. 2010;
7: 1171–1180https://doi.org/10.1098/rsif.2009.0559 Crossref | Scopus (84) | Google ScholarSee all References. However, pure ceramic scaffolds are usually brittle and prone to fracture which hampers their application in large, load-bearing defects22x22Ostrowski, N, Roy, A, and Kumta, PN. Magnesium Phosphate Cement Systems for Hard Tissue Applications: A Review. ACS Biomater Sci Eng. 2016;
2: 1067–1083https://doi.org/10.1021/acsbiomaterials.6b00056 Crossref | Scopus (123) | Google ScholarSee all References. A previous study has investigated a ceramic-polymer composite of MgPSr and polycaprolactone (PCL), which is a versatile biomaterial ink that can be processed through extrusion-based 3D printing at room temperature. The biomaterial can be manufactured into different complex geometries to improve bone filling of a defect without compromising mechanical stability23x23Golafshan, N, Vorndran, E, Zaharievski, S et al. Tough magnesium phosphate-based 3D-printed implants induce bone regeneration in an equine defect model. Biomaterials. 2020;
261: 120302https://doi.org/10.1016/j.biomaterials.2020.120302 Crossref | Scopus (59) | Google ScholarSee all References. The porous nature of a printed osteotomy wedge scaffold allows bone marrow to populate the wedge upon implantation. Alternatively, pre-surgical seeding of the implant with a bioactive product accelerating osteogenesis, such as bone marrow concentrate (BMC), could further accelerate bone union.
This study aimed to design and manufacture a scaffold as gap filler in OWO around the knee joint. The mechanical stability of the wedge scaffold, as well as the in vitro osteoinductive properties of the material on MSCs and BMC were investigated. Additionally, preservation of the pre-designed implant structure and height were assessed upon implantation into human cadaveric legs.
Materials and Methods
Study outline
To completely fill the opening wedge gap after an OWO, 3D printed scaffolds were manufactured using patient computed tomography (CT) data and computer-aided design. The printed scaffold structures were mechanically characterized and in vitro potency of the MgPSr-PCL material was evaluated to induce osteogenesis when seeded with bone marrow-derived MSCs, as well as BMC. Finally, a proof-of-concept surgical implantation in a cadaver model was performed for implementation of the implants into the current osteotomy procedure.
Computer-aided design of osteotomy wedge
For initial design of a wedge scaffold for mechanical characterization and in vitro experiments, an anonymized CT scan and surgical planning for an 8 mm medial opening-wedge distal femur osteotomy was acquired from a clinical case (University Medical Center Utrecht) (Figure 1Ai). The computer-aided design (CAD) of the wedge scaffold was developed in SolidWorks software (Dassault Systèmes, Waltham, MA, USA), using the CT scan images. After assessment of the wedge scaffold, BioCAM™ software was used to define the wedge scaffold internal architecture and subsequently translate the design into a G-Code. The external wall of the osteotomy scaffold was kept closed with two outer layers, while for the internal region of the osteotomy scaffolds, three different inter-fibre spacings (IFS), 1.3 mm, 1.0 mm, and 0.7 mm (abbreviated as IFS-1.3, IFS-1.0, and IFS-0.7, respectively) were considered.
Material preparation and extrusion-based 3D-bioprinting
The biomaterial ink was prepared by combining in-house synthesized Mg2.33Sr0.67(PO4)2 powder and commercial medical grade poly(ε-caprolactone) (mPCL, Purasorb PC 12, Purac Biomaterials, Netherlands) in a weight ratio of 70:30 wt.% of MgPSr to PCL, according to a procedure previously described23x23Golafshan, N, Vorndran, E, Zaharievski, S et al. Tough magnesium phosphate-based 3D-printed implants induce bone regeneration in an equine defect model. Biomaterials. 2020;
261: 120302https://doi.org/10.1016/j.biomaterials.2020.120302 Crossref | Scopus (59) | Google ScholarSee all References. Designed scaffolds were fabricated by an extrusion-based 3D-printing system (3D Discovery, regenHU, Switzerland) using the MgPSr-PCL biomaterial ink. The ink was transferred to a 10 mL syringe (Nordson EFD, USA) and extruded though a 22G conical nozzle (inner diameter = 0.41 mm, Nordson EFD, USA) at a pressure of 0.9 bar and collected at collector speed of 6 mm/s.
Mechanical characterization of printed wedge scaffolds
Uniaxial compression tests were performed using a universal testing machine (Zwick Z010, Germany) equipped with a 1 kN load cell. Tests were performed on cylindrical samples (d = 6 mm, h = 12 mm, n = 5) for all three groups (IFS-1.3, IFS-1.0 and IFS-0.7, without closed outer edges), at a rate of 1 mm/min (at room temperature). From the engineered stress-strain curves, the elastic modulus (defined as the slope of the linear region at the interval 0.02 - 0.05 mm/mm strain), the yield stress (defined as the point where nonlinear deformation begins), and toughness (defined as the absorbed energy by the scaffolds up to yield stress) were determined.
In vitro accelerated degradation of printed wedge scaffolds
The degradation of the materials was studied under controlled conditions which accelerated biomaterial degradation in vitro22x22Ostrowski, N, Roy, A, and Kumta, PN. Magnesium Phosphate Cement Systems for Hard Tissue Applications: A Review. ACS Biomater Sci Eng. 2016;
2: 1067–1083https://doi.org/10.1021/acsbiomaterials.6b00056 Crossref | Scopus (123) | Google ScholarSee all References. Wedge scaffolds were incubated in a 0.4 mg/ml lipase solution (from Pseudomonas cepacia, Sigma-Aldrich) and 1 mg/ml sodium azide (Sigma-Aldrich) at 37ºC for 15 days. At each time point (1, 5, 10, and 15 days), the enzymatic solution was refreshed, and samples were monitored for weight loss, quantified as follows:
(1)
In vitro osteogenesis of scaffolds
Donors and cell isolation
Human bone marrow-derived mesenchymal stromal cells (BM-MSCs) were derived from healthy donor bone marrow aspirates (n = 3, age range 2 – 12) as approved by the Dutch central Committee on Research Involving Human Subjects (CCMO, Bio-banking bone marrow for MSC expansion, NL41015.041.12). The parent or legal guardian of the donor signed the informed consent approved by the CCMO. In brief, the mononuclear fraction was separated using a density gradient (Lympoprep, Axis Shield). MSCs were isolated by plastic adherence and expanded for three passages in Minimum Essential Media (αMEM, Macopharma) with 5% platelet lysate and 3.3 IU/mL heparin and cryopreserved. Subsequently, MSCs were expanded for two additional passages in MSC expansion medium (αMEM [Gibco], 10% (v/v) fetal bovine serum [FBS; Biowest], 1% penicillin/streptomycin [pen/strep; 100 U/mL, 100 µg/mL], 200 µM l-ascorbic acid 2-phosphate [ASAP; Sigma-Aldrich], and 1 ng/mL basic fibroblast growth factor [bFGF; PeproTech]). BMC was obtained from donors undergoing an OWO or total knee arthroplasty surgery (n = 2, age range 39 - 49) after their informed consent (protocol approved by the local medical ethical committee). Bone marrow was concentrated to one tenth of its original volume using Ficoll paque (GE Healthcare) density separation.
In vitro culture of scaffolds
Standardized 5 mm diameter cylindrical scaffolds (ISF-1.0) were printed as described before23x23Golafshan, N, Vorndran, E, Zaharievski, S et al. Tough magnesium phosphate-based 3D-printed implants induce bone regeneration in an equine defect model. Biomaterials. 2020;
261: 120302https://doi.org/10.1016/j.biomaterials.2020.120302 Crossref | Scopus (59) | Google ScholarSee all References and sterilized by washing in 70% ethanol and Milli-Q, followed by exposure to ultraviolet light for 1 hour. Scaffolds were cut in half with a sterile scalpel and seeded with either 15,000 MSCs / scaffold in fibrin gel (25 µL fibrinogen [1:15 in PBS] crosslinked with 25 µL thrombin (1:50 in PBS); Tisseel, Baxter) or 25 µL BMC (crosslinked with 16.6 µL thrombin and 16.6 µL CaCl2 [500 mM in 0.9% NaCl]). Cell-seeded scaffolds were pre-cultured in MSC expansion medium for two days, followed by 21 days of osteogenic induction with osteogenic differentiation medium (αMEM supplemented with 10% FBS, 1% pen/strep, 200 µM ASAP, 10 mM β-glycerophosphate [Sigma-Aldrich], and 10 nM dexamethasone [Sigma-Aldrich]). Control cell-seeded scaffolds were treated with MSC expansion medium without bFGF.
Alkaline phosphatase, calcium, and DNA quantification
Osteogenic differentiation of the cells was measured by the activity of the early osteogenic marker alkaline phosphatase (ALP) after 5, 7, and 11 days and by quantification of calcium produced after 21 days. To determine activity of ALP, cells were lysed in Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) by three freeze-thaw cycles. ALP activity was measured using the conversion of p-nitrophenyl phosphate liquid substrate (pNPP, Sigma-Aldrich). Absorbance was measured every minute for 30 minutes at 405 nm and corrected for absorbance at 655 nm. Calf intestinal ALP (Sigma-Aldrich) was used as a standard. Calcium concentration in the samples was quantified after 21 days using a colorimetric calcium assay kit (Abcam) according to the manufacturer's instructions. ALP activity and calcium levels were corrected for DNA. DNA content was determined using the Quant-iT PicoGreen dsDNA assay (Invitrogen) according to the manufacturer's instructions.
Osteocalcin immunocytochemistry
To visualise the osteogenic marker osteocalcin, scaffolds were fixed in formalin for 30 minutes for the osteocalcin immunocytochemistry after 21 days of differentiation. Samples were permeabilized with 0.2% (v/v) Triton X-100 in phosphate-buffered saline (PBS), followed by blocking with 5% (v/v) bovine serum albumin (BSA) in PBS. Next, samples were incubated overnight at 4ºC with 10 µg/mL mouse-anti-human primary antibody against osteocalcin (clone OCG4; Enzo Life Sciences). Samples were then incubated with 10 µg/mL goat-anti-mouse antibody conjugated to Alex Fluor 488 (Invitrogen) for one hour at room temperature. All samples were also stained for F-actin (1:200; phalloidin-TRITC; Sigma-Aldrich) and 4′,6-diamidino-2-phenylindole (100 ng/mL; DAPI; Sigma-Aldrich). Images were acquired with a Leica SP8X Laser Scanning Confocal Microscope and Leica LASX acquisition software.
Ex vivo surgical implantation of the printed wedges
Three fresh-frozen human cadaveric legs were obtained (all left legs, one male and two female) in accordance with the guidelines of the local medical ethical committee. CT-scans were obtained of the three included legs (Philips Healthcare, Best, The Netherlands; 100 kV and 130mAs), with 0.8 mm slice thickness. Single plane OWOs were pre-operatively planned in 3-Matic (Materialise, Leuven, Belgium), with for each leg a specific wedge height (5, 10, and 15 mm). This resulted in post-surgical 3D-models of the cadavers with left open osteotomy gaps, which functioned as surrogate for the 3D printing of the wedges. A proximal biplanar medial high tibial OWO was performed following a standard surgery protocol. During this procedure, the osteotomy gap was kept open using a lamina spreader and the 3D-printed scaffold wedge was inserted into the gap. The tips of the wedge scaffolds were resected to fit the biplanar osteotomy gap, without altering the outside rim of the wedge. The osteotomies were then fixated with angular stable plates (Activmotion, Newclip Technics, Haute-Goulaine, France). The same plates are used for patients in the clinic and have a smaller footprint compared to other commercially used plates. Following implantation of the scaffold wedges, additional CT scans were obtained of the operated cadaver legs, subsequently the wedges were explanted for further analysis.
Micro-computed tomography
The pre- and post-surgical wedge scaffolds underwent micro-CT (Quantum FX-Perkin Elmer, USA) for height analyses. Scan parameters were 90 kV tube voltage, 180 µA tube current, 60 or 73 mm resolution, and 2 min scan time. Scaffold heights pre- and post-implantation were quantified using computer vision software Fiji (software version 2.1.0/1.53c, National Institutes of Health, Bethesda, USA). Scaffold height was measured at the highest point. The micro-CT images of a single scaffold pre- and post-implantation were superimposed to ensure the scaffolds were measured at the same location at both timepoints.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 8.3 (GraphPad Software, Inc., La Jolla, CA, USA). All data were presented as mean ± standard deviation (SD). To test for differences in mechanical evaluations and calcium content, a one-way analysis of variance (ANOVA) with Tukey's post hoc test was used. To test for differences in DNA and ALP quantifications, a two-way ANOVA with Tukey's post hoc was used. For scaffold wedge height, a two-tailored t-test was used. Normality was confirmed with a Shapiro-Wilk test (p>0.05). P values below 0.05 were considered significant.
Results
Personalized implant design and fabrication
Wedge implants were designed for both open-wedge lateral distal femur (for in vitro analyses; Figure 1AFigure 1A) and medial tibial osteotomies (for implantation in cadavers; Figure 1BFigure 1B) from CT scan 3D reconstructions (Panels i). Wedges had closed outer edges, aimed at limiting leakage from the osteotomy site into the soft tissues surrounding the bone, while the interior was porous (Panels ii and iii).
Figure 1
Surgical planning and extrusion-based printing. Wedge design for open-wedge osteotomies in (A) distal femur and (B) proximal tibia. (Panels i) Surgical planning of open-wedge osteotomies derived from computed tomography (CT) scans. (Panels ii) Top views of printing paths of computer-aided designs (CAD) of personalized wedge implants. (Panels iii) The finalized personalized wedges in magnesium strontium phosphate-polycaprolactone (MgPSr-PCL) biomaterial. Scale bar = 10 mm.
Incorporation of the thermoplastic PCL into the ceramic MgPSr phase improved handling of the implants, overcoming downsides of brittle ceramic materials22x22Ostrowski, N, Roy, A, and Kumta, PN. Magnesium Phosphate Cement Systems for Hard Tissue Applications: A Review. ACS Biomater Sci Eng. 2016;
2: 1067–1083https://doi.org/10.1021/acsbiomaterials.6b00056 Crossref | Scopus (123) | Google ScholarSee all References. The stress-strain curves of the standardized printed discs with different IFS showed comparable profiles (Figure 2AFigure 2A). The decrease in IFS resulted in an increase in mechanical stability, elastic modulus increased significantly from 105.3 ± 10.26 MPa (IFS-1.3) to 151.5 ± 12.61 MPa (IFS-0.7) (Figure 2CFigure 2C). Yield stress, defined as the point of maximum elastic deformation, increased from 4.2 ± 1.27 MPa (IFS-1.3) to 6.0 ± 1.90 MPa and 11.4 ± 1.86 MPa for IFS-1.0 and IFS-0.7, respectively (Figure 2DFigure 2D). In line, strain energy increased from 0.077 ± 0.0208 J for IFS-1.3 to 0.196 ± 0.0957 J for IFS-0.7 (Figure 2EFigure 2E). While a disc with an IFS of 0.7 mm presented the highest elastic modulus, printing of a complete wedge scaffold with this IFS resulted in a construct that was not completely porous from top to bottom, which is essential to flow of bone marrow through the scaffold in vivo (Figure 2BFigure 2B). Scaffolds with a planned IFS of 1.0 mm resulted in completely porous wedges and only a slight difference in elastic modulus and strain energy compared to scaffolds with a planned IFS of 0.7 mm, which did not reach statistical significance. While the degradation rate of IFS-1.3 samples (as evaluated under accelerated degradation conditions) was 30% faster compared to IFS-1.0 samples (Figure 2FFigure 2F), the combination of tested characteristics led to the selection of IFS-1.0 for further analyses as the best compromise between open porosity, mechanical stability, and degradation properties.
Figure 2
Evaluation of mechanical properties of the printed magnesium strontium phosphate-polycaprolactone (MgPSr-PCL) wedges. A) Longitudinal compression profile of 3D printed MgPSr-PCL wedge scaffolds for inter-fibre spacing (IFS) -1.3, IFS-1.0, and IFS-0.7. B) Corresponding photographs showed the different scaffolds after the printing. Open pores in wedges IFS-1.3 and IFS-1.0 can be appreciated, while IFS-0.7 wedges were not porous. C) Elastic modulus, D) Yield stress, and E) strain energy from compressive loading profile for IFS-1.3, IFS-1.0, and IFS-0.7. F) Weight loss of wedge scaffolds during accelerated in vitro degradation in enzymatic solution over 15 days. *p<0.05
In vitro osteogenic properties of the scaffold material
Human BM-MSCs embedded in fibrin (MSC-fibrin) were seeded in the biomaterial scaffolds to evaluate osteogenic potential. Additionally, to simulate the in vivo situation, a second group of scaffolds was seeded with BMC. The culture-expanded human MSCs attached to the MgPSr-PCL material and proliferated over time. Cells in BMC also proliferated on the scaffolds (Figure 4AFigure 4A and Supplemental Figure 1). Activity of the early osteogenic marker ALP was similar in MSC-fibrin and BMC groups when scaffolds were cultured in control medium, yet were increased in MSC-fibrin when cultured in osteogenic medium (Figure 4BFigure 4B). BMC performed similar to MSC-fibrin in terms of calcium production at 21 days of culture (Figure 4CFigure 4C). Of note, both experimental groups had a higher ALP activity and increased calcium production compared to MSCs that were cultured in monolayers, indicating osteoconductive effects of the scaffold material and 3D environment. Production of osteocalcin, an exclusive marker for osteoblasts, was observed in cultures under all conditions irrespective of culture medium used (Figure 3DFigure 3D).
Figure 3
In vitro osteogenic performance of the magnesium strontium phosphate-polycaprolactone (MgPSr-PCL) biomaterial. A) Quantification of DNA in the MgPSr-PCL scaffolds at 5, 7, 11, and 21 days in culture with control (left panel) and osteogenic medium (right panel). B) Early osteogenic marker alkaline phosphatase (ALP) activity relative to the amount of DNA at 5, 7, and 11 days. C) Calcium content of the cultured constructs at day 21 in control medium (top panel) and osteogenic medium (bottom panel). D) Immunocytochemical osteocalcin staining on 21-day cultured standardized cylindrical MgPSr-PCL scaffolds (ISF-1.0) using: (i) culture expanded mesenchymal stromal cells (MSC) in control medium, (ii) MSC in osteogenic medium, (iii) bone marrow concentrate (BMC) in control medium, and (iv) BMC in osteogenic medium. Nuclei are shown in blue (DAPI), osteocalcin expression in green, and F-actin in red. Dashed lines indicate the location of three-dimensional scaffold material. Scale bar = 100 µm. *p<0.05
Three fresh-frozen human cadaveric legs underwent CT scanning in order to plan three OWOs with different heights; 5, 10, and 15 mm (Figure 4AFigure 4A, 4B4B, 4C4C). 3D models of the tibias were used to plan single plane osteotomy gap (Panels i) and design the wedge scaffolds (Panels ii). Wedges were implanted during a standard proximal biplanar OWO procedure and fixated with an angular stable plate (Panels iii). Post-surgical X rays (Panels iv) and CT scans (Panels v) illustrate the scaffold positioning and fit. Micro-CT analyses of the wedges pre- and post-implantation indicated good analogy of the scaffolds (Figure 5AFigure 5A, pre-operative in red, post-operative in grey). Due to the biplanar approach of the osteotomy procedure24x24Lobenhoffer, P and Agneskirchner, JD. Improvements in surgical technique of valgus high tibial osteotomy. Knee Surgery, Sports Traumatology, Arthroscopy. 2003;
11: 132–138https://doi.org/10.1007/s00167-002-0334-7 Crossref | PubMed | Scopus (433) | Google ScholarSee all References, the scaffolds were adjusted at one side using an automatic saw, not altering the rest of the scaffold shape and outer rim (Figure 5BFigure 5B, arrows indicating trimmed edge). Quantification of wedge heights from micro-CT images revealed that the wedge heights were not affected by the applied surgical procedure, during which they bear loading for a brief moment when the laminar spreader is removed to allow sufficient space for the plate to be fixated (Figure 5CFigure 5C).
Figure 4
Surgical implantation of personalized scaffold wedges. Planned osteotomy heights of A) 5 mm, B) 10 mm, and C) 15 mm from computed tomography (CT) scans of human cadaveric legs (Panels i). (Panels ii) 3D printed wedge scaffolds in magnesium strontium phosphate-polycaprolactone (MgPSr-PCL). (Panels iii) Scaffolds implanted in the cadaveric legs. (Panels iv) X-ray of the legs after implantation. (Panels v) 3D reconstruction from CT scans after implantation of the wedge scaffolds.
Micro-computed tomography (CT) analysis of printed wedge scaffolds. A) 3D reconstructions of the wedges from micro-CT images of the printed wedges pre- and post-implantation. Pre-implanted scaffolds in red, post-implanted scaffolds in grey. B) Micro-CT reconstruction of the 5 mm wedge. Note the small portion that was adjusted during the procedure (indicated by the white arrows). C) Quantification of scaffold wedge height before and after implantation into the cadaveric legs revealed no loss of scaffold height.
This study demonstrated a proof of concept to manufacture implants for bone gap filling in femoral or tibial OWOs, by 3D printing a biodegradable and osteoinductive scaffold material. The printed material promoted osteogenesis of MSCs and BMC in vitro and scaffolds were implanted ex vivo without compromising pre-operatively planned wedge height. The aim was to design implants that fitted the planned osteotomy gap and height, fabricate these, and evaluate their implementation in the established surgical procedure.
Post-surgical pain poses a challenge in OWO care. One of the hypotheses for the cause of pain is bleeding from the osteotomy site. It has been shown that fitting an allogeneic graft, which closes the gap completely, enables early postoperative weight-bearing and improve clinical outcomes after three months8x8van Genechten, W, van den Bempt, M, van Tilborg, W et al. Structural allograft impaction enables fast rehabilitation in opening-wedge high tibial osteotomy: a consecutive case series with one year follow-up. Knee Surgery, Sports Traumatology, Arthroscopy. 2020;
28: 3747–3757https://doi.org/10.1007/s00167-019-05765-z Crossref | Scopus (8) | Google ScholarSee all References. A variety of scaffold materials and growth factors is being investigated to accelerate osteogenesis in OWO. Most studies investigating a scaffold material make use of wedges composed of hydroxyapatite or beta-tricalcium phosphate7x7Han, JH, Kim, HJ, Song, JG et al. Is Bone Grafting Necessary in Opening Wedge High Tibial Osteotomy? A Meta-Analysis of Radiological Outcomes. Knee Surg Relat Res. 2015;
27: 207–220https://doi.org/10.5792/ksrr.2015.27.4.207 Crossref | Scopus (33) | Google ScholarSee all References,14x14Hooper, NM, Schouten, R, and Hooper, GJ. The Outcome of Bone Substitute Wedges in Medial Opening High Tibial Osteotomy. Open Orthop J. 2013;
7: 373–377https://doi.org/10.2174/1874325001307010373 Crossref | Google ScholarSee all References,25x25Sasaki, S, Maeyama, A, Kiyama, T et al. Combined use of beta-tricalcium phosphate with different porosities can accelerate bone remodelling in open-wedge high tibial osteotomy. Asia Pac J Sports Med Arthrosc Rehabil Technol. 2022;
29: 30–34https://doi.org/10.1016/j.asmart.2022.05.004 Crossref | Scopus (0) | Google ScholarSee all References, whereas some focus on injection of biologics, like platelet-rich plasma and MSCs26x26Koh, YG, Kwon, OR, Kim, YS, and Choi, YJ. Comparative outcomes of open-wedge high tibial osteotomy with platelet-rich plasma alone or in combination with mesenchymal stem cell treatment: A prospective study. Arthroscopy - Journal of Arthroscopic and Related Surgery. 2014;
30: 1453–1460https://doi.org/10.1016/j.arthro.2014.05.036 Abstract | Full Text | Full Text PDF | PubMed | Scopus (148) | Google ScholarSee all References, or use a single growth factor, like bone morphogenetic protein (BMP) 627x27Chiari, C, Grgurevic, L, Bordukalo-Niksic, T et al. Recombinant Human BMP6 Applied Within Autologous Blood Coagulum Accelerates Bone Healing: Randomized Controlled Trial in High Tibial Osteotomy Patients. Journal of Bone and Mineral Research. 2020;
35: 1893–1903https://doi.org/10.1002/jbmr.4107 Crossref | Scopus (20) | Google ScholarSee all References.
While a great deal of effort is put into accelerating bone healing, the current study investigates a combined approach of osteoinduction and filling of the osteotomy gap. The presented methods offer the possibility of personalizing an osteoinductive wedge implant and incorporating this into the existing 3D workflow. Yet, the manufactured implants were designed to match opening-wedge height and the anatomy of the bone. Adjustment of the scaffold edge enabled proper fit into the osteotomy plane, while still filling the gap. Precisely sealing of the whole gap was proven to be challenging in the current setup. The approach used in the current study led to a relatively symmetrical wedge, as opposed to a trapezoid-shaped wedge. To move towards clinical implementation and complete sealing of an osteotomy gap, a more precise, biplanar surgical planning should be performed. Most likely, this would also require using 3D printed patient-specific instruments (PSI), with pre-operatively determined saw cuts resulting in a predefined gap morphology, for which a fitting implant can subsequently be fabricated. In some cases of malalignment correction, PSI are preferred by orthopaedic surgeons, in the form of saw and drill guides per-operatively. Further research can offer the possibility of adding a personalized wedge implant into this workflow. With predetermined bone cuts and gap morphology, our wedge scaffold can be 3D printed pre-operatively to fit. However, this initial study focussed primarily on the feasibility of implanting a 3D printed MgPSr-PCL wedge scaffold, without compromising the pre-operatively planned wedge height.
While most of the load on the osteotomy gap is absorbed by the angular stable plate and screws, the implant should remain stable during the brief period before the plate is fixed on the bone. The elastic modulus of the implants demonstrated similarities to human trabecular bone28x28Gerhardt, LC and Boccaccini, AR. Bioactive glass and glass-ceramic scaffolds for bone tissue engineering. Materials. 2010;
3: 3867–3910https://doi.org/10.3390/ma3073867 Crossref | Scopus (773) | Google ScholarSee all References. Scaffolds with a planned fibre spacing of 1.0 mm maintained open pores, through which bone marrow would be allowed to flow and osteogenesis might be accelerated. While implant height was maintained post-implantation in the cadaveric legs, future in vivo and clinical studies are necessary to confirm maintenance of pre-planned wedge height during a longer period of implantation, as well as speed of bone union.
The full-size implants degraded over time in an accelerated in vitro setup using an enzymatic solution. Prior research in an equine model has shown that degradation of the MgPSr-PCL (pore size 1.0 mm) in the enzymatic solution for ten days corresponded to degradation over six months during in vivo implantation23x23Golafshan, N, Vorndran, E, Zaharievski, S et al. Tough magnesium phosphate-based 3D-printed implants induce bone regeneration in an equine defect model. Biomaterials. 2020;
261: 120302https://doi.org/10.1016/j.biomaterials.2020.120302 Crossref | Scopus (59) | Google ScholarSee all References. Increasing inter-fibre spacing accelerated degradation by 30% in mass loss, indicating that in vivo mass loss might also be accelerated. However, the exact degradation and speed of bone formation of the osteotomy-specific MgPSr-PCL implants in this specific anatomical location should be evaluated in a large animal model.
The printed MgPSr-PCL material facilitated osteogenesis in vitro, confirming previous findings23x23Golafshan, N, Vorndran, E, Zaharievski, S et al. Tough magnesium phosphate-based 3D-printed implants induce bone regeneration in an equine defect model. Biomaterials. 2020;
261: 120302https://doi.org/10.1016/j.biomaterials.2020.120302 Crossref | Scopus (59) | Google ScholarSee all References. For large defects, pre-seeding of the scaffold with a regenerative compound, like MSCs, might be beneficial to accelerate bone healing. Here, seeding of the material with both culture-expanded MSCs and BMC resulted in production of osteoblast-specific markers, indicating that infiltrated bone marrow in the scaffold material after implantation may be sufficient by itself to stimulate osteogenesis. This is most likely induced by its growth factor-rich nature and the presence of progenitor cells29x29Fortier, LA, Strauss, EJ, Shepard, DO, Becktell, L, and Kennedy, JG. Biological Effects of Bone Marrow Concentrate in Knee Pathologies. Journal of Knee Surgery. 2019;
32: 2–8https://doi.org/10.1055/s-0038-1676069 Crossref | PubMed | Scopus (23) | Google ScholarSee all References. While fabrication of personalized 3D implants from patient imaging data was demonstrated before for orthopaedic applications30x30Willemsen, K, Nizak, R, Noordmans, HJ, Castelein, RM, Weinans, H, and Kruyt, MC. Challenges in the design and regulatory approval of 3D-printed surgical implants: a two-case series. Lancet Digit Health. 2019;
1: e163–e171https://doi.org/10.1016/S2589-7500(19)30067-6 Abstract | Full Text | Full Text PDF | Scopus (57) | Google ScholarSee all References,31x31Willemsen, K, Tryfonidou, M, Sakkers, R et al. Patient-specific 3D-printed shelf implant for the treatment of hip dysplasia: Anatomical and biomechanical outcomes in a canine model. Journal of Orthopaedic Research. 2021;
: 1–9https://doi.org/10.1002/jor.25133 Crossref | Scopus (6) | Google ScholarSee all References, this study is the first to report on a personalized biodegradable implant for OWO.
The current study was mainly limited by the imbalance between preoperative osteotomy planning (single plane) and the intra-operative surgical biplanar osteotomy, leading to a slight mismatch between gap and fabricated implant. By implementing 3D printed PSI in the workflow, an optimal fit of the designed implant can be achieved. Because the purpose of this study was to design and manufacture a gap-filling wedge implant in an osteoinductive material and to evaluate this in an ex vivo model, achieving a perfect fit was beyond the scope of this investigation.
Conclusions
To conclude, a gap-filling implant for open-wedge osteotomies was designed and manufactured. This implant was 3D manufactured in an osteoinductive and biodegradable material that supported cell attachment, growth, and production of early and late osteogenic markers in vitro. Finally, an ex vivo proof-of-concept of the surgical procedure was successfully performed, implementing the designed wedge scaffolds into the standard osteotomy procedure, while maintaining implant integrity and pre-planned wedge height.
Author's Contributions
LV, NvE, RC, and JM conceived the study. All authors were involved in design of the study. MR, NG, and MdR carried out the laboratory experiments. NG and MC prepared the biomaterial ink. MR, HN, NvE, and RC were involved in the cadaver study. MR, HN, and NG drafted the manuscript. All authors revised the manuscript critically and have given final approval of the version to be published.
Supplemental Figure 1. DNA quantification. Quantification of DNA in scaffolds seeded with bone marrow-derived mesenchymal stromal cells (MSC) and bone marrow concentrate (BMC) compared to MSCs cultured in monolayers as controls.
Declaration of Competing Interest
Nienke van Egmond reports a relationship with Newclip Technics that includes: consulting or advisory.
Lucienne Vonk reports a relationship with CO.DON AG that includes: employment.
Lucienne Vonk reports a relationship with International Cartilage Regeneration & Joint Preservation Society that includes: non-financial support.
Senior Associate Editor for The Journal of Cartilage and Joint Preservation - L.V.
Editor for The Journal of Cartilage and Joint Preservation - R.C.
Editor for The Journal of Cartilage and Joint Preservation - J.M.
Funding
This research is supported by the partners of Regenerative Medicine Crossing Borders (RegMed XB), financed by the Dutch Ministry of Economic Affairs by means of the PPP Allowance made available by the Top Sector Life Sciences & Health to stimulate public-private partnership, and ReumaNederland ( LLP-12, LLP22 , and 19-1-207 MINIJOINT ). JM and MC acknowledge financial support from the Gravitation Program “Materials Driven Regeneration”, funded by the Netherlands Organization for Scientific Research ( 024.003.013 ). MC also acknowledges funding from Reprint project ( OCENW.XS5. 161 ) by Netherlands Organization Scientific Research.
Acknowledgements
The authors would like to thank Arno Mooring (iMoveMedical, Nieuwegein, The Netherlands) for providing surgical tools and the 3D Lab of the University Medical Center for their assistance in planning of the osteotomies. The authors would also like to thank Nils van Veen for assisting with the CT scans and Marco Rondhuis for his kind assistance at the Anatomy Department. The authors also thank Dr. Elke Vorndran and Prof. Uwe Gbureck from the Department for Functional Materials in Medicine and Dentistry, University of Würzburg, for kindly synthetizing and providing the ceramic biomaterial used in this work.
Informed Patient Consent
This research did not include any human subjects and therefore no written consent was necessary to be obtained.
1Appel, H and Friberg, S. The Effect of High Tibial Osteotomy on Pain in Osteoarthritis of the Knee Joint. Acta Orthop. 1972;
43: 558–565https://doi.org/10.3109/17453677208991278
2Ekhtiari, S, Haldane, CE, de Sa, D, Simunovic, N, Musahl, V, and Ayeni, OR. Return to work and sport following high tibial osteotomy: A systematic review. Journal of Bone and Joint Surgery - American Volume. 2016;
98: 1568–1577https://doi.org/10.2106/JBJS.16.00036
3Lobenhoffer, P. Indication for Unicompartmental Knee Replacement versus Osteotomy around the Knee. Journal of Knee Surgery. 2017;
30: 769–773https://doi.org/10.1055/s-0037-1605558
4van Haeringen, MH, Kuijer, PPFM, Daams, JG et al. Opening- and closing-wedge high tibial osteotomy are comparable and early full weight bearing is safe with angular stable plate fixation: a meta-analysis. (Published online)Knee Surgery, Sports Traumatology, Arthroscopy. 2022;
https://doi.org/10.1007/s00167-022-07229-3
5Wang, Z, Zeng, Y, She, W, Luo, X, and Cai, L. Is opening-wedge high tibial osteotomy superior to closing-wedge high tibial osteotomy in treatment of unicompartmental osteoarthritis? A meta-analysis of randomized controlled trials. International Journal of Surgery. 2018;
60: 153–163https://doi.org/10.1016/j.ijsu.2018.10.045
7Han, JH, Kim, HJ, Song, JG et al. Is Bone Grafting Necessary in Opening Wedge High Tibial Osteotomy? A Meta-Analysis of Radiological Outcomes. Knee Surg Relat Res. 2015;
27: 207–220https://doi.org/10.5792/ksrr.2015.27.4.207
8van Genechten, W, van den Bempt, M, van Tilborg, W et al. Structural allograft impaction enables fast rehabilitation in opening-wedge high tibial osteotomy: a consecutive case series with one year follow-up. Knee Surgery, Sports Traumatology, Arthroscopy. 2020;
28: 3747–3757https://doi.org/10.1007/s00167-019-05765-z
9Cao, ZW, Mai, XJ, Wang, J, Feng, EH, and Huang, YM. Uni-compartmental knee arthroplasty versus high tibial osteotomy for knee osteoarthritis: a systematic review and meta-analysis. Journal of Arthroplasty. 2018;
33: 952–959https://doi.org/10.1016/j.arth.2017.10.025
11Lee, JS, Park, YJ, Wang, L, Chang, YS, Shetty, GM, and Nha, KW. Modified Iliac Crest Reconstruction with Bone Cement for Reduction of Donor Site Pain and Morbidity after Open Wedge High Tibial Osteotomy: A Prospective Study. Knee Surg Relat Res. 2016;
28: 277–282https://doi.org/10.5792/ksrr.15.046
12Kuremsky, MA, Schaller, TM, Hall, CC, Roehr, BA, and Masonis, JL. Comparison of autograft vs allograft in opening-wedge high tibial osteotomy. Journal of Arthroplasty. 2010;
25: 951–957https://doi.org/10.1016/j.arth.2009.07.026
13Lode, A, Meissner, K, Luo, Y et al. Fabrication of porous scaffolds by three-dimensional plotting of a pasty calcium phosphate bone cement under mild conditions. J Tissue Eng Regen Med. 2014;
8: 682–693https://doi.org/10.1002/term.1563
14Hooper, NM, Schouten, R, and Hooper, GJ. The Outcome of Bone Substitute Wedges in Medial Opening High Tibial Osteotomy. Open Orthop J. 2013;
7: 373–377https://doi.org/10.2174/1874325001307010373
15Castilho, M, Moseke, C, Ewald, A et al. Direct 3D powder printing of biphasic calcium phosphate scaffolds for substitution of complex bone defects. Biofabrication. 2014;
6https://doi.org/10.1088/1758-5082/6/1/015006
16Yang, X, Xie, B, Wang, L, Qin, Y, Henneman, ZJ, and Nancollas, GH. Influence of magnesium ions and amino acids on the nucleation and growth of hydroxyapatite. CrystEngComm. 2011;
13: 1153–1158https://doi.org/10.1039/c0ce00470g
17Yang, F, Yang, D, Tu, J, Zheng, Q, Cai, L, and Wang, L. Strontium enhances osteogenic differentiation of mesenchymal stem cells and in vivo bone formation by activating Wnt/catenin signaling. Stem Cells. 2011;
29: 981–991https://doi.org/10.1002/stem.646
18Reitmaier, S, Kovtun, A, Schuelke, J et al. Strontium(II) and mechanical loading additively augment bone formation in calcium phosphate scaffolds. Journal of Orthopaedic Research. 2018;
36: 106–117https://doi.org/10.1002/jor.23623
19Lode, A, Heiss, C, Knapp, G et al. Strontium-modified premixed calcium phosphate cements for the therapy of osteoporotic bone defects. Acta Biomater. 2018;
65: 475–485https://doi.org/10.1016/j.actbio.2017.10.036
20Kim, JA, Lim, J, Naren, R, suk, Yun H, and Park, EK. Effect of the biodegradation rate controlled by pore structures in magnesium phosphate ceramic scaffolds on bone tissue regeneration in vivo. Acta Biomater. 2016;
44: 155–167https://doi.org/10.1016/j.actbio.2016.08.039
21Jia, J, Zhou, H, Wei, J et al. Development of magnesium calcium phosphate biocement for bone regeneration. J R Soc Interface. 2010;
7: 1171–1180https://doi.org/10.1098/rsif.2009.0559
22Ostrowski, N, Roy, A, and Kumta, PN. Magnesium Phosphate Cement Systems for Hard Tissue Applications: A Review. ACS Biomater Sci Eng. 2016;
2: 1067–1083https://doi.org/10.1021/acsbiomaterials.6b00056
23Golafshan, N, Vorndran, E, Zaharievski, S et al. Tough magnesium phosphate-based 3D-printed implants induce bone regeneration in an equine defect model. Biomaterials. 2020;
261: 120302https://doi.org/10.1016/j.biomaterials.2020.120302
24Lobenhoffer, P and Agneskirchner, JD. Improvements in surgical technique of valgus high tibial osteotomy. Knee Surgery, Sports Traumatology, Arthroscopy. 2003;
11: 132–138https://doi.org/10.1007/s00167-002-0334-7
25Sasaki, S, Maeyama, A, Kiyama, T et al. Combined use of beta-tricalcium phosphate with different porosities can accelerate bone remodelling in open-wedge high tibial osteotomy. Asia Pac J Sports Med Arthrosc Rehabil Technol. 2022;
29: 30–34https://doi.org/10.1016/j.asmart.2022.05.004
26Koh, YG, Kwon, OR, Kim, YS, and Choi, YJ. Comparative outcomes of open-wedge high tibial osteotomy with platelet-rich plasma alone or in combination with mesenchymal stem cell treatment: A prospective study. Arthroscopy - Journal of Arthroscopic and Related Surgery. 2014;
30: 1453–1460https://doi.org/10.1016/j.arthro.2014.05.036
27Chiari, C, Grgurevic, L, Bordukalo-Niksic, T et al. Recombinant Human BMP6 Applied Within Autologous Blood Coagulum Accelerates Bone Healing: Randomized Controlled Trial in High Tibial Osteotomy Patients. Journal of Bone and Mineral Research. 2020;
35: 1893–1903https://doi.org/10.1002/jbmr.4107
28Gerhardt, LC and Boccaccini, AR. Bioactive glass and glass-ceramic scaffolds for bone tissue engineering. Materials. 2010;
3: 3867–3910https://doi.org/10.3390/ma3073867
31Willemsen, K, Tryfonidou, M, Sakkers, R et al. Patient-specific 3D-printed shelf implant for the treatment of hip dysplasia: Anatomical and biomechanical outcomes in a canine model. Journal of Orthopaedic Research. 2021;
: 1–9https://doi.org/10.1002/jor.25133
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