Preparation of a biphasic scaffold for osteochondral tissue engineering

Tissue engineering has been developed as a prospective approach for the repair of articular cartilage defects. Engineered osteochondral implants can facilitate the fixation and integration with host tissue, and therefore promote the regeneration of osteochondral defects. A biphasic scaffold with a stratified two-layer structure for osteochondral tissue engineering was developed from biodegradable synthetic and naturally derived polymers. The upper layer of the scaffold for cartilage engineering was collagen sponge; the lower layer for bone engineering was a composite sponge of poly(DL-lactic-co-glycolic acid) (PLGA) and naturally derived collagen. The PLGA–collagen composite sponge layer had a composite structure with collagen microsponge formed in the pores of a skeleton PLGA sponge. The collagen sponge in the two respective layers was connected. Observation of the collagen/PLGA–collagen biphasic scaffold by scanning electron microscopy (SEM) demonstrated the connected stratified structure. The biphasic scaffold was used for culture of canine bone-marrow-derived mesenchymal stem cells. The cell/scaffold construct was implanted in an osteochondral defect in the knee of a one-year old beagle. Osteochondral tissue was regenerated four months after implantation. Cartilage- and bone-like tissues were formed in the respective layers. The collagen/PLGA–collagen biphasic scaffold will be useful for osteochondral tissue engineering.     

Evaluation of a press-fit osteochondral poly(ester-urethane) scaffold in a rabbit defect model

The purpose of this study was to evaluate the impact on osteochondral healing of press-fitted multiphasic osteochondral scaffolds consisting of poly(ester-urethane) (PUR) and hydroxyapatite into a cylindric osteochondral defect in the distal non-weight bearing femoral trochlear ridge of the rabbit. Two scaffolds were investigated, one with and one without an intermediate microporous membrane between the cartilage and the bone compartment of the scaffold. A control group without a scaffold placed into the defect was included. After 12 weeks macroscopic and histomorphological analyses were performed. The scaffold was easily press-fitted and provided a stable matrix for tissue repair. The membrane did not demonstrate a detrimental effect on tissue healing compared with the scaffold without membrane. However, the control group had statistically superior healing as reflected by histological differences in the cartilage and subchondral bone compartment between control group and each scaffold group. A more detailed analysis revealed that the difference was localized in the bone compartment healing. The present study demonstrates that an elastomeric PUR scaffold can easily be press-fitted into an osteochondral defect and provides a stable matrix for tissue repair. However, the multi-phasic scaffold did not provide a clear advantage for tissue healing. Future investigations should refine especially the bone phase of the implant to increase its stiffness, biocompatibility and osteoconductive activity. A more precise fabrication technique would be necessary for the matching of tissue organisation.     

Novel alginate biphasic scaffold for osteochondral regeneration: an in vivo evaluation in rabbit and sheep models

Current therapeutic strategies for osteochondral restoration showed a limited regenerative potential. In fact, to promote the growth of articular cartilage and subchondral bone is a real challenge, due to the different functional and anatomical properties. To this purpose, alginate is a promising biomaterial for a scaffold-based approach, claiming optimal biocompatibility and good chondrogenic potential. A previously developed mineralized alginate scaffold was investigated in terms of the ability to support osteochondral regeneration both in a large and medium size animal model. The results were evaluated macroscopically and by microtomography, histology, histomorphometry, and immunohistochemical analysis. No evidence of adverse or inflammatory reactions was observed in both models, but limited subchondral bone formation was present, together with a slow scaffold resorption time.The implantation of this biphasic alginate scaffold provided partial osteochondral regeneration in the animal model. Further studies are needed to evaluate possible improvement in terms of osteochondral tissue regeneration for this biomaterial.     

A novel osteochondral scaffold of ceramic–gelatin assembly for articular cartilage repair

A novel ceramic–gelatin assembly (CGA) has been designed as an osteochondral scaffold for articular cartilage repair. The CGA scaffold consists of four layers, that is, a porous ceramic layer as osseous component and also as anchor, a dense ceramic layer to prevent blood vessel penetration and also to stand shear stress, a porous ceramic layer for fixation of bone to cartilage, i.e. for joining the ceramic part to the porous gelatin layer, the latter being used as cartilaginous component. The joining was done by the infiltration of gelatin solution into the porous ceramic layer, gelling and crosslinking. This CGA scaffold can offer solutions to the so-far not satisfactorily resolved issues of the osteochondral scaffold, i.e. anchoring, blood vessel penetration, shear stress distribution during articular joint motion, and enough strength to join the cartilaginous component to the osseous component to prevent delamination. This novel scaffold was tested by in vitro cell culture with Wistar rat's joint chondrocytes. DNA assay, GAGs assay, RT-PCR, and histological evaluations with hematoxylin–eosin and Safranin-O staining were carried out to show that cartilage tissue can be developed in four weeks.     

骨软骨组织工程仿生梯度支架研究进展

骨软骨缺损是导致关节发病和残疾的重要原因,骨软骨组织工程是修复骨软骨缺损的方法之一。骨软骨组织工程方法涉及仿生梯度支架的制造,该支架需模仿天然骨软骨组织的生理特性（例如从软骨表面到软骨下骨之间的梯度过渡）。在许多研究中骨软骨仿生梯度支架表现为离散梯度或连续梯度,用于模仿骨软骨组织的特性,例如生物化学组成、结构和力学性能。连续型骨软骨梯度支架的优点是其每层之间没有明显的界面,因此更相似地模拟天然骨软骨组织。到目前为止,骨软骨仿生梯度支架在骨软骨缺损修复研究中已经取得了良好的实验结果,但是骨软骨仿生梯度支架与天然骨软骨组织之间仍然存在差异,其临床应用还需要进一步研究。本文首先从骨软骨缺损的背景、微尺度结构与力学性能、骨软骨仿生梯度支架制造相关的材料与方法等方面概述了离散和连续梯度支架的研究进展。其次,由于3D打印骨软骨仿生梯度支架的方法能够精确控制支架孔的几何形状和力学性能,因此进一步介绍了计算仿真模型在骨软骨组织工程中的应用,例如采用仿真模型优化支架结构和力学性能以预测组织再生。最后,提出了骨软骨缺损修复相关的挑战以及骨软骨组织再生未来研究的展望。例如,连续型骨软骨仿生梯度支架需要更相似地模拟天然骨软骨组织单元的结构,即力学性能和生化性能的过渡更加自然地平滑。同时,虽然大多数骨软骨仿生梯度支架在体内外实验中均取得了良好的效果,但临床研究和应用仍然需要进行进一步深入研究。     

Fabrication and characterization of a biphasic scaffold for osteochondral tissue engineering

A biphasic scaffold with a stratified structure for osteochondral tissue engineering was developed. The chondral phase was a collagen-chitosan composite. The osseous phase was a composite of bioactive glass and collagen. Collagen integrated in the two respective phases was connected by cross-linking agents. Both layers of the scaffold showed interconnected porous structures. After being immersed into stimulated body fluid, precipitation of spherulitic grains could be found on the surface of the osseous phase and this precipitation was proved to be hydroxyapatite by X-ray diffraction and Fourier transform infrared spectroscopy. Inversion, fluorescence and scanning electron microscopy further confirmed that bone marrow stromal cells could anchor on this scaffold with healthy spreading. As the consequence, this biphasic scaffold may have significant potential as an alternative for osteochondral tissue engineering.     

Bi-layered constructs based on poly(l-lactic acid) and starch for tissue engineering of osteochondral defects

Articular cartilage has a limited capacity to repair itself, and conventional therapeutic approaches have shown to have limited success as they are deficient and inconsistent in long-term repair. Tissue engineering has shown to be an alternative route to regenerate articular defects. In this work, new bi-layered scaffolds are developed in order to enhance the integration between the engineered cartilage tissue and the corresponding subchondral bone. The concept includes the use of a common polymer in both sides, poly(l-lactic acid), PLLA, to increase the bonding between them, and the use of compression moulding followed by particle leaching to process porous scaffolds with controllable porosities. A compact layer could be observed between the two layers that could be useful for independent cell culturing of the developed osteochondral constructs. A blend of starch and PLLA was used in the cartilage side, which was found to possess adequate hydration capability. For the bone region, where more stiffness and strength was required, PLLA reinforced with hydroxyapatite was used. Preliminary bioactivity tests demonstrated that the bone-layer could induce the formation of a calcium–phosphate layer in vitro, whereas the cartilage layer does not exhibit the ability for calcification.     

A Three‐Layered Osteochondral Plug: Structural,Mechanical, and in vitro Biocompatibility Analysis

This study aims to produce an osteochondral plug with three distinct layers resembling the naturally occurring cartilage, tidemark, and subchondral zones, for the regeneration of defects of articular cartilage. The bone layer is constructed from a PLLA/PCL polymeric blend using a dual‐porogen approach. The pore surfaces are coated with type‐I collagen and hydroxyapatite. The upper layer, made of PGA nonwoven felt, is combined with the lower using a polymeric blend with a pigment for better visualization during implantation. Vertical channels are formed from the bottom layer to the upper border of the tidemark to facilitate the delivery of stem cells and blood from the bone marrow when implanted. The Young's modulus of the osteochondral plugs is 94.5 ± 9.42 kPa. Cell‐culture studies confirm the biocompatibility.     

Characterization of the repair tissue in articular cartilage defects using silver-enhanced colloidal gold immunostaining

The newly developed silver-enhanced colloidal gold staining method was used in a rabbit model to characterize the repair tissue in large articular cartilage defects filled with a heterocyclic methacrylate polymer. By 6 weeks the resurfacing tissue consisted of highly organized hyaline-like articular cartilage, fully integrated with the adjacent normal cartilage. Immuno-histochemistry detected collagen type ll, keratan sulphate, chondroitin 4-sulphate and chondroitin 6-sulphate in the matrix of the neocartilage. The level to which the polymer plug was recessed apeared to be critical to the overall quality of the repair tissue. Optimum results were obtained when the top surface of the biomaterial was at the level of the subchondral bone, below the level of the surrounding articular cartilage. Other technical aspects of implantation, that also affect the repair, are also discussed.     

The regeneration of articular cartilage using a new polymer system

A polymer system based on room temperature polymerising poly (ethylmethacrylate) polymer powder and tetrahydrofurfuryl monomer has been investigated as a biomaterial for encouraging articular cartilage repair. This heterocyclic methacrylate polymer system swells slightly in situ and thus provides a good interface with subchondral bone resulting in mechanical stability with favourable uptake kinetics. Another feature of this polymer system is that it exhibits high water uptake which leads to absorption of the surrounding tissue fluid and matrix proteins, including growth factors; this may encourage the formation of new cartilage. Three weeks after implantation the tissue overgrowth contained cartilage components: chondrocytes, collagen type II, chondroitin 4-sulphate and chondroitin 6-sulphate. In addition numerous chondrocyte clones were observed at the edge of the defect and in the newly repaired tissue. By six weeks a superficial articulating surface was continuous with the normal articular cartilage with underlying tissue which showed some evidence of endochondral ossification. By nine weeks the surface covering of new cartilage had a widened and an irregular zone of calcified cartilage with thickened subchondral bone was present. At eight months the resurfaced cartilage remained intact above a remodelled subchondral bone end plate.     

聚氨酯软骨-骨双层复合材料的构建及性能研究

关节软骨损伤是临床上的常见病,由于其组织再生能力差,可能导致骨性关节炎的发生,因此,研究开发骨-软骨移植替代材料非常重要。目的就是设计一体化软骨-骨双层复合材料,以解决软骨与骨的整合问题。该双层复合体上层软骨材料为聚氨酯,软骨下骨为羟基磷灰石/聚氨酯复合支架材料,两层结构中引用了同一种材料——聚氨酯,将双层结构有机黏合在一起,使黏合更牢固。下层多孔HA/PU复合支架材料的孔与孔之间相互贯通,孔隙率约为83%,孔径范围分布在200～600μm。体外细胞相容性实验表明,该一体化双层复合材料为细胞的黏附、增殖以及生存活力的维持提供了有利环境。上述结果表明该双层复合材料有望用于软骨组织工程修复。     

Osteochondral tissue engineering using a PLGA–collagen hybrid mesh

Canine bone marrow stromal cells were cultured in a PLGA–collagen hybrid mesh in osteogenic medium in vitro and laminated to construct an osteo layer. Canine articular chondrocytes were cultured in the hybrid mesh in DMEM containing 10% FBS and laminated to construct a chondral layer. The osteo and chondral layers were sutured together and implanted subcutaneously in nude mice. The original round disc shape of the osteochondral constructs was preserved during the implantation. The osteo and chondral layers appeared red and glistening white, respectively. Histological examination of the implant specimens indicated that stromal cells and chondrocytes were evenly distributed throughout the scaffold. The laminated meshes were bound together and the two layers had a distinct interface between them. The cells showed a round morphology in the chondral layer and a spindle morphology in the osteo layer. In the chondral layer, spherical chondrocytes were surrounded by an abundant cartilaginous extracellular matrix. The round morphology and positive stain by safranin-O and toluidine blue, together with the expression of genes encoding type II collagen and aggrecan suggested the formation of neocartilage in the chondral layer. Expressions of genes encoding type I collagen and osteocalcin were detected in the osteochondral implant. These results indicate the formation of osteochondral-like tissue, and the hybrid mesh and lamination method may be useful for osteochondral tissue engineering.     

A hydrophylic polymer system enhanced articular cartilage regeneration in vivo

This study describes a new method for the repair of large articular cartilage defects in the knee joint and compares the effect of two polymer systems on the quality of the repair tissue. The two systems are a newly developed hydrophylic system, based on poly-ethyl-methacrylate (PEMA) polymer and tetra-hydro-furfuryl-methacrylate (THFMA) monomer and the conventional bone cement polymer system, based on poly-methyl-methacrylate (PMMA) polymer and methyl-methacrylate (MMA) monomer. Thirty adult Sandy-lop rabbits were used. Both knees were operated on in each animal, the one defect received either PEMA/THFMA or conventional bone cement and the contralateral defect received no biomaterial (control group). Femora were retrieved at six weeks and the repair tissue was studied by histology, histochemistry and immuno-histochemistry. PEMA/THFMA enhanced the quality of the repair significantly (p<0.0001). By six weeks hyaline-like articular cartilage was the predominant tissue covering the defects and it was fully integrated with the surrounding normal articular cartilage. Immuno-localization showed cartilage components, including collagen type II, distributed evenly throughout its matrix. PMMA/MMA on the other hand did not improve significantly the repair tissue, which was predominately fibro-cartilaginous, poorly bonded to the adjacent normal articular cartilage. The method of implantation is simple and easily reproducible and the new polymer has been well-accepted by the rabbits.     

Combination of bone marrow concentrate and PGA scaffolds enhance bone marrow stimulation in rabbit articular cartilage repair

Bone marrow stimulation (BMS) has been regarded as a first-line procedure for the repair of articular cartilage. However, cartilage repair using BMS alone has so far not been ideal because cell homing to the required area has not been sufficient. The aim of this study was to investigate the feasibility of autologous bone marrow concentrate transplantation for the repair of large, full-thickness cartilage defects. Thirty rabbits were divided into five groups: untreated (control); BMS only (BMS); BMS followed by PGA implantation (PGA); BMS followed by a combination of PGA and autologous bone marrow concentrate (BMC); and BMS together with a composite of PGA and cultured bone marrow stem cells (BME). The animals were sacrificed at week 8 after operation, and HE staining, toluidine blue staining and immunohistochemistry were used to assess the repair of defects. The results showed that improved repair, including more newly formed cartilage tissue and hyaline cartilage-specific extracellular matrix, was observed in BMC group relative to the first three groups, in addition similar results were found between BMC and BME groups, however it took longer time for in vitro cell expansion in the BME group. This study demonstrates that the transplantation of autologous bone marrow concentrate is an easy, safe and potentially viable method to contribute to articular cartilage repair.     

A one-step treatment for chondral and osteochondral knee defects: clinical results of a biomimetic scaffold implantation at 2 years of follow-up

The increasing interest in the role of subchondral bone with regard to articular surface disease led to the development of new bioengineered strategies. Aim of this study is to evaluate the clinical and MRI outcome after the implantation of a nanostructured biomimetic three-phasic collagen–hydroxyapatite construct for the treatment of chondral and osteochondral defects of the knee in a large cohort of patients. Seventy-nine patients (63 M, 16 W), affected by grade III–IV femoral condyle or trochlea chondral lesions or osteochondritis dissecans (OCD) were consecutively treated. Mean age was 31.0 ± 11.3 years, mean lesion size was 3.2 ± 2.0 cm². Fifty patients underwent previous surgeries, concurrent procedures were necessary in 39 cases. The clinical outcome was evaluated using the IKDC and Tegner scores at 12 and 24 months of follow-up. At follow-up times an MRI was performed and evaluated with the MOCART score. All the scores improved significantly from the baseline. IKDC subjective score showed a further increase between 12 and 24 months of follow-up, and 82.2 % of the patients improved their symptoms at the final evaluation. Patients affected by OCDs had better results than those with degenerative lesions. Some abnormal MRI findings were present, even though no correlation was found with the clinical outcome. This one-step biomimetic approach developed to favor osteochondral tissue regeneration is effective in treating knees affected by damages of the articular surface, leading to a significant clinical improvement. However, abnormal MRI findings were present, even if not correlated with the clinical outcome.     

3D bioprinting of multicellular scaffolds for osteochondral regeneration

Regeneration of osteochondral tissue is of great potentialities in repairing severe osteochondral defects. However, anisotropic physiological characteristics and tissue linage difference make the regeneration of osteochondral tissue remain a huge challenge. Herein, a multicellular system based on a bilayered co-culture scaffold mimicking osteochondral tissues was successfully developed for an alternative of osteochondral regeneration via a 3D bioprinting strategy. The dual function of integrally repairing both cartilage and bone could be achieved by designing multiple-cells distribution and a cell-induced bioink containing bioceramic particles. As an important bioactive agent, the Li-Mg-Si bioceramics-containing bioink exhibited the function of simultaneously stimulating multiple cells for differentiation towards specific directions. The 3D bioprinted co-culture scaffolds showed the capacity for osteochondral tissue regeneration by inducing osteogenic and chondrogenic differentiation in vitro and accelerating the repair of severe osteochondral defects in vivo. This study offers a potential strategy for complex tissue reconstruction through bioprinting multiple tissue cells in combination of bioceramics-stimulating bioinks.     

A silk fibroin/chitosan scaffold in combination with bone marrow-derived mesenchymal stem cells to repair cartilage defects in the rabbit knee

Bone marrow-derived mesenchymal stem cells (BMSCs) were seeded in a three-dimensional scaffold of silk fibroin (SF) and chitosan (CS) to repair cartilage defects in the rabbit knee. Totally 54 rabbits were randomly assigned to BMSCs + SF/CS scaffold, SF/CS scaffold and control groups. A cylindrical defect was created at the patellofemoral facet of the right knee of each rabbit and repaired by scaffold respectively. Samples were prepared at 4, 8 and 12 weeks post-surgery for gross observation, hematoxylin–eosin and toluidine blue staining, type II collagen immunohistochemistry, Wakitani histology. The results showed that differentiated BMSCs proliferated well in the scaffold. In the BMSCs + SF/CS scaffold group, the bone defect was nearly repaired, the scaffold was absorbed and immunohistochemistry was positive. In the SF/CS scaffold alone group, fiber-like tissues were observed, the scaffold was nearly degraded and immunohistochemistry was weakly positive. In the control group, the defect was not well repaired and positive immunoreactions were not detected. Modified Wakitani scores were superior in the BMSCs + SF/CS scaffold group compared with those in other groups at 4, 8 and 12 weeks (P < 0.05). A SF/CS scaffold can serve as carrier for stem cells to repair cartilage defects and may be used for cartilage tissue engineering.     

Enhanced repair of a critical-sized segmental bone defect in rabbit femur by surface microstructured porous titanium

Repair of load-bearing bone defects remains a challenge in the field of orthopaedic surgery. In the current study, a surface microstructured porous titanium (STPT) successively treated with H₂O₂/TaCl₅ solution and simulated body fluid was used to repair the critical-sized segmental bone defects in rabbit femur, and non-treated porous titanium (NTPT) and porous biphasic calcium phosphate ceramics (PBCP) were used as control, respectively. A 15 mm long implant was positioned in the femoral defect and stabilized by a plate and screws fixation. After implantation into the body for 1, 3 and 6 months, X-ray observation confirmed that porous titanium groups (NTPT and STPT) provided better mechanical support than PBCP group at the early stage. However, there was no obvious difference in the formed bony callus between PBCP and STPT groups in the later stage, and they both showed better shape of bony callus than NTPT group. Micro-CT and histomorphometric analysis for the samples of 6-month implantation demonstrated that more new bone formed in the inner pores of PBCP and STPT groups than that in NTPT group. Moreover, the biomechanical tests revealed that STPT group could bear larger compressive load than NTPT and PBCP groups, almost reaching the level of the normal rabbit femur. STPT exhibited the enhanced repairing effect on the critical-sized segmental bone defect in rabbit femur, meaning that it could be an ideal material for the repair of large bone defect in load-bearing site.     

A simple method of cartilage regeneration using a new polymerizing system: ultrastructural characteristics of the repair tissue

The ultrastructural characteristics of the repair tissue in large articular cartilage defects, filled with a heterocyclic polymerizing system were studied using transmission electron microscopy (TEM) and energy dispersive micro-analysis (EDMA). By six weeks post-implantation, the defects were resurfaced with predominantly hyaline-like articular cartilage. Chondrocytes in both the superficial and deep zones of the repair tissue were highly productive, secreting large amounts of proteoglycans, into a well-organized, rich in collagen fibrils, extracellular matrix. By contrast, in the repair tissue of the defects treated without the biomaterial, proteoglycan synthesis was less and the structure of the matrix was inferior. We conclude that the polymer enhances both chondrocyte metabolism and matrix organization, thus improving the quality of the repair tissue in articular cartilage defects.