Implementation of Photosensitive, Injectable, Interpenetrating, and Kartogenin-Modified GELMA/PEDGA Biomimetic Scaffolds to Restore Cartilage Integrity in a Full-Thickness Osteochondral Defect Model.

Cartilage defects caused by mechanical tear and wear are challenging clinical problems. Articular cartilage has unique load-bearing properties and limited self-repair ability. The current treatment methods, such as microfractures and autogenous cartilage transplantation to repair full-thickness cartilage defects, have apparent limitations. Tissue engineering technology has the potential to repair cartilage defects and directs current research development. To enhance the regenerative capacities of cartilage in weight-bearing areas, we attempted to develop a biomimetic scaffold loaded with a chondroprotective factor that can recreate structure, restore mechanical properties, and facilitate anabolic metabolism in larger joint defects. For enhanced spatial control over both bone and cartilage layers, it is envisioned that biomaterials that meet the needs of both tissue components are required for successful osteochondral repair. We used gelatin methacrylate (GELMA) and polyethylene glycol diacrylate (PEGDA) light-cured dual-network cross-linking modes that can significantly increase the mechanical properties of scaffolds and are capable of restoring function and prolonging the degradation time. Once the hydrogel complex was injected into the osteochondral defect, in situ UV light curing was applied to seamlessly connect the defect repair tissue with the surrounding normal cartilage tissue. The small molecule active substance kartogenin (KGN) can promote cartilage repair. We encapsulated KGN in biomimetic scaffolds so that, as the scaffold degrades, scaffold-loaded KGN was slowly released to induce endogenous mesenchymal stem cells to home and differentiate into chondrocytes to repair defective cartilage tissue. Our experiments have proven that, compared with the control group, GELMA/PEGDA + KGN repaired cartilage defects and restored cartilage to hyaline cartilage. Our study suggests that implementing photosensitive, injectable, interpenetrating, and kartogenin-modified GELMA/PEDGA biomimetic scaffolds may be a novel approach to restore cartilage integrity in full-thickness osteochondral defects.

[Design & fabrication of porous core implant with preset channel network for osteonecrosis of the femoral head].

Referring to the anatomical characterization of natural spongy bone and channel network in cortical bone, we designed a new pattern of biomimetic impalnt with preset channel for blood vessel inserting to treat early femoral head necrosis. The surgical ptrocedure was simulated by CAD model. Ceramic stereolithography was applied to fabricate the green part. Other process, such as dehydration, rinsing, drying and sintering, were taken successively. The final ceramic part kept identical with the engineered part either in the shape or in the internal structure. No deformation or crack happened. Pore size, interconnected pore size, porosity and interconnected porosity of ceramic part could satisfy cellular grouth. Spectrum analysis showed that no phase transition or chemical reaction happened during fabrication process. The biocompatibility of the final part kept the same with original during beta-TCP powder. The compressive strength was 23.54 MPa, close to natural spongy bone. It is an ideal implant to treat early femoral head necroseis because it makes preimplantation of cells and biological factors, blood velssel inserting, early establishment of blood supply possible. At the same time, it could provide enough mechanical support to prevent collapse of femoral head. It could provide a wide clinical foreground.

Implanting hydroxyapatite-coated porous titanium with bone morphogenetic protein-2 and hyaluronic acid into distal femoral metaphysis of rabbits.

OBJECTIVE: To assess the osseointegration capability of hydroxyapatite-coated porous titanium with bone morphogenetic protein-2 (BMP-2) and hyaluronic acid to repair defects in the distal femur metaphysis in rabbits. METHODS: Porous titanium implants were made by sintering titanium powder at high temperature, which were coated with hydroxyapatite by alkali and heat treatment and with BMP-2 combined with bone regeneration materials. And hyaluronic acid was further used as delivery system to prolong the effect of BMP-2. The implants were inserted into the metaphysis of the distal femur of rabbits. The animals were killed at 6, 12 and 24 weeks to accomplish histological and biomechanical analyses. RESULTS: According to the result of histological analysis, the osseointegration in BMP-2 group was better than that of the HA-coated porous titanium group. In push-out test, all the samples had bigger shear stress as time passed by. There was statistical difference between the two groups in 6 and 12 weeks but not in 24 weeks. CONCLUSION: Hydroxyapatite-coated porous titanium with BMP-2 and hyaluronic acid has a good effect in repairing defects of distal femur in rabbits, which is a fine biotechnology for future clinical application.

The effect of interface microstructure on interfacial shear strength for osteochondral scaffolds based on biomimetic design and 3D printing.

Interface integration between chondral phase and osseous phase is crucial in engineered osteochondral scaffolds. However, the integration was poorly understood and commonly failed to meet the need of osteochondral scaffolds. In this paper, a biphasic polyethylene glycol (PEG)/ß-tricalcium phosphate (ß-TCP) scaffold with enhanced interfacial integration was developed. The chondral phase was a PEG hydrogel. The osseous phase was a ß-TCP ceramic scaffold. The PEG hydrogel was directly cured on the ceramic interface layer by layer to fabricate osteochondral scaffolds by 3D printing technology. Meanwhile, a series of interface structure were designed with different interface pore area percentages (0/10/20/30/40/50/60%), and interfacial shear test was applied for interface structure optimization (n=6 samples/group). The interfacial shear strength of 30% pore area group was nearly three folds improved compared with that of 0% pore area percentage group, and more than fifty folds improved compared with that of traditional integration (5.91±0.59 kPa). In conclusion, the biomimetic PEG/ß-TCP scaffolds with interface structure enhanced integration show promising potential application for osteochondral tissue engineering.

[Fabrication and in vivo implantation of ligament-bone composite scaffolds based on three-dimensional printing technique].

OBJECTIVE: To solve the fixation problem between ligament grafts and host bones in ligament reconstruction surgery by using ligament-bone composite scaffolds to repair the ligaments, to explore the fabrication method for ligament-bone composite scaffolds based on three-dimensional (3-D) printing technique, and to investigate their mechanical and biological properties in animal experiments. METHODS: The model of bone scaffolds was designed using CAD software, and the corresponding negative mould was created by boolean operation. 3-D printing techinique was employed to fabricate resin mold. Ceramic bone scaffolds were obtained by casting the ceramic slurry in the resin mould and sintering the dried ceramics-resin composites. Ligament scaffolds were obtained by weaving degummed silk fibers, and then assembled with bone scaffolds and bone anchors. The resultant ligament-bone composite scaffolds were implanted into 10 porcine left anterior cruciate ligament rupture models at the age of 4 months. Mechanical testing and histological examination were performed at 3 months postoperatively, and natural anterior cruciate ligaments of the right sides served as control. RESULTS: Biomechanical testing showed that the natural anterior cruciate ligament of control group can withstand maximum tensile force of (1 384 +/- 181) N and dynamic creep of (0.74 +/- 0.21) mm, while the regenerated ligament-bone scaffolds of experimental group can withstand maximum tensile force of (370 +/- 103) N and dynamic creep of (1.48 +/- 0.49) mm, showing significant differences (t = 11.617, P = 0.000; t = 2.991, P = 0.020). In experimental group, histological examination showed that new bone formed in bone scaffolds. A hierarchical transition structure regenerated between ligament-bone scaffolds and the host bones, which was similar to the structural organizations of natural ligament-bone interface. CONCLUSION: Ligament-bone composite scaffolds based on 3-D printing technique facilitates the regeneration of biomimetic ligament-bone interface. It is expected to achieve physical fixation between ligament grafts and host bone.

A novel silk-TCP-PEEK construct for anterior cruciate ligament reconstruction: an off-the shelf alternative to a bone-tendon-bone autograft.

Bone-tendon-bone autograft represents a gold-standard for anterior cruciate ligament (ACL) reconstruction but at the cost of a secondary surgical site that can be accompanied by functional impairment and discomfort. Although numerous in vitro and in vivo studies have investigated tissue engineering alternatives to autografting, the achievement of a functional histological transition between soft and hard tissue has remained elusive. To bridge this gap we developed and tested a novel multiphase scaffold of silk, tricalcium phosphate (TCP) and polyether ether ketone for ACL reconstruction. We present in vitro biomechanical tests demonstrating that the construct recapitulates native ACL function under typical physiological loads. A pilot in vivo experiment in two pigs with a three-month follow-up showed a robust histological transition between regenerated fibrous tissue and the margins of the bone tunnel, with histological features similar to the native ACL to bone insertion. These histological observations suggest that the construct was stably anchored until TCP incorporation to the host tissues. On the strength of these preliminary results, we conclude that the described approach may offer a promising alternative to autograft for ACL reconstruction. This study thus provides proof for a concept that warrants further development.

A novel silk-based artificial ligament and tricalcium phosphate/polyether ether ketone anchor for anterior cruciate ligament reconstruction - safety and efficacy in a porcine model.

Loss of ligament graft tension in early postoperative stages following anterior cruciate ligament (ACL) reconstruction can come from a variety of factors, with slow graft integration to bone being widely viewed as a chief culprit. Toward an off-the-shelf ACL graft that can rapidly integrate to host tissue, we have developed a silk-based ACL graft combined with a tricalcium phosphate (TCP)/polyether ether ketone anchor. In the present study we tested the safety and efficacy of this concept in a porcine model, with postoperative assessments at 3months (n=10) and 6months (n=4). Biomechanical tests were performed after euthanization, with ultimate tensile strengths at 3months of ∼370N and at 6months of ∼566N - comparable to autograft and allograft performance in this animal model. Comprehensive histological observations revealed that TCP substantially enhanced silk graft to bone attachment. Interdigitation of soft and hard tissues was observed, with regenerated fibrocartilage characterizing a transitional zone from silk graft to bone that was similar to native ligament bone attachments. We conclude that both initial stability and robust long-term biological attachment were consistently achieved using the tested construct, supporting a large potential for silk-TCP combinations in the repair of the torn ACL.

Cartilage repair and subchondral bone migration using 3D printing osteochondral composites: a one-year-period study in rabbit trochlea.

Increasing evidences show that subchondral bone may play a significant role in the repair or progression of cartilage damage in situ. However, the exact change of subchondral bone during osteochondral repair is still poorly understood. In this paper, biphasic osteochondral composite scaffolds were fabricated by 3D printing technology using PEG hydrogel and ß-TCP ceramic and then implanted in rabbit trochlea within a critical size defect model. Animals were euthanized at 1, 2, 4, 8, 16, 24, and 52 weeks after implantation. Histological results showed that hyaline-like cartilage formed along with white smooth surface and invisible margin at 24 weeks postoperatively, typical tidemark formation at 52 weeks. The repaired subchondral bone formed from 16 to 52 weeks in a "flow like" manner from surrounding bone to the defect center gradually. Statistical analysis illustrated that both subchondral bone volume and migration area percentage were highly correlated with the gross appearance Wayne score of repaired cartilage. Therefore, subchondral bone migration is related to cartilage repair for critical size osteochondral defects. Furthermore, the subchondral bone remodeling proceeds in a "flow like" manner and repaired cartilage with tidemark implies that the biphasic PEG/ß-TCP composites fabricated by 3D printing provides a feasible strategy for osteochondral tissue engineering application.

[Cartilage repair and subchondral bone reconstruction based on three-dimensional printing technique].

OBJECTIVE: To investigate whether subchondral bone microstructural parameters are related to cartilage repair during large osteochondral defect repairing based on three-dimensional (3-D) printing technique. METHODS: Biomimetic biphasic osteochondral composite scaffolds were fabricated by using 3-D printing technique. The right trochlea critical sized defects (4.8 mm in diameter, 7.5 mm in depth) were created in 40 New Zealand white rabbits (aged 6 months, weighing 2.5-3.5 kg). Biomimetic biphasic osteochondral composite scaffolds were implanted into the defects in the experimental group (n = 35), and no composite scaffolds implantation served as control group (n = 5); the left side had no defect as sham-operation group. Animals of experimental and sham-operation groups were euthanized at 1, 2, 4, 8, 16, 24, and 52 weeks after operation, while animals of control group were sampled at 24 weeks. Subchondral bone microstructural parameters and cartilage repair were quantitatively analyzed using Micro-CT and Wayne scoring system. Correlation analysis and regression analysis were applied to reveal the relationship between subchondral bone parameters and cartilage repair. The subchondral bone parameters included bone volume fraction (BV/TV), bone surface area fraction (BSA/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular spacing (Tb.Sp). RESULTS: In the experimental group, articular cartilage repair was significantly improved at 52 weeks postoperatively, which was dominated by hyaline cartilage tissue, and tidal line formed. Wayne scores at 24 and 52 weeks were significantly higher than that at 16 weeks in the experimental group (P < 0.05), but no significant difference was found between at 24 and 52 weeks (P > 0.05); the scores of experimental group were significantly lower than those of sham-operation group at all time points (P < 0.05). In the experimental group, new subchondral bone migrated from the surrounding defect to the centre, and subchondral bony plate formed at 24 and 52 weeks. The microstructural parameters of repaired subchondral bone followed a "twin peaks" like discipline to which BV/TV, BSA/BV, and Tb.N increased at 2 and 16 weeks, and then they returned to normal level. The Tb.Sp showed reversed discipline compared to the former 3 parameters, no significant change was found for Tb.Th during the repair process. Correlation analysis showed that BV/TV, BSA/BV, Tb.Th, Tb.N, and Tb.Sp were all related with gross appearance score and histology score of repaired cartilage. CONCLUSION: Subchondral bone parameters are related with cartilage repair in critical size osteochondral repair in vivo. Microstructural parameters of repaired subchondral bone follow a "twin peaks" like discipline (osteoplasia-remodeling-osteoplasia-remodeling) to achieve reconstruction, 2nd week and 16th week are critical time points for subchondral bone functional restoration.

Design and fabrication of a novel porous implant with pre-set channels based on ceramic stereolithography for vascular implantation.

Being a multi-etiological factors disease, osteonecrosis of the femoral head affects many young people, leading to the collapse of the femur head; eventually the hip arthroplasty is needed if not treated in time. Unfortunately, as yet, no satisfactory therapy to repair necrotic bone at an early stage is present. Novel implants with pre-set channels were designed for the treatment of early femoral head necrosis. Ceramic stereolithography was applied to fabricate the green part from ß-TCP powder. Other processes, such as dehydration, rinsing, drying and sintering, were processed successively. The final ceramic part remains the same as the engineered part in both shape and internal structure. No significant deformation or crack occurred. X-ray diffraction showed that no facies changed or chemical reaction occurred during the fabrication process. The chemical composition remains the same as that of the original ß-TCP powder. The compressive strength is 23.54 MPa, close to that of natural cancellous bone. Novel implants with a pre-set channel were designed and fabricated for blood vessel implantation. Bioceramic stereolithography technology based directly on the CAD model in this research shows advantages in accurate design, optimization of 3D scaffold and critical control of the fabrication process. This proposed implant shows promising clinical application in the restoration of early femoral head necrosis.