Repair of osteochondral defect with tissue-engineered two-phase composite material of injectable calcium phosphate and hyaluronan sponge

Articular cartilage has limited capacity for repair. In the present study, tissue-engineered two-phase composite material was used for the repair of osteochondral defects in young adult rabbit knee. This composite material is composed of an injectable calcium phosphate (ICP) and a hyaluronan (HA) derivate of either ACP or HYAFF 11 sponge. The osteochondral defect, 3 mm in diameter and 3 mm deep, was created in the weight-bearing region of the medial femoral condyle. The bone portion of the defect was first filled with ICP to a level approximately 1 mm below the articular surface. HA sponge (3 mm in diameter and 1-1.2 mm thick), with or without loading of autologous bone marrow-derived progenitor cells (MPCs), was then inserted into the defect on top of the ICP as it hardened. Animals were allowed free cage activity postoperatively, and killed 4 or 12 weeks (for the HYAFF 11 sponge group) after the surgery. At 4 weeks, histological examination showed that the defect was filled up to 90-100% of its depth. Whitish repair tissue on the top appeared to be integrated with the surrounding articular cartilage. Four distinct zones of repair tissue were identified: a superficial layer, a chondroid tissue layer, an interface between HA sponge and ICP, and the ICP material. Evidence of extensive osteoclastic and osteoblastic activities was observed in the bone tissue surrounding the defect edge and in ICP material. By 12 weeks, the zonal features of the repair tissue became more distinct; chondrocytes were arranged in a columnar array, and a calcified layer of cartilage was formed beneath the chondroid tissue in some specimens. The healing tissue of the HA sponge material loaded with MPCs had higher cellular density and better integration with the surrounding cartilage than HA sponge material not loaded with MPCs. This study suggests that using a two-phase composite graft may hold potential for the repair of osteochondral defects by providing mechanical support that mimicks subchondral bone, while also providing a chondrogenic scaffold for the top cartilage repair.     

Bilayered scaffolds for osteochondral tissue engineering

Osteoarthritis (OA) is a prevalent degenerative joint disease that places a significant burden on the socioeconomic efficacy of communities around the world. Tissue engineering repair of articular cartilage in synovial joints represents a potential OA treatment strategy superior to current surgical techniques. In particular, osteochondral tissue engineering, which promotes the simultaneous regeneration of articular cartilage and underlining subchondral bone, may be a clinically relevant approach toward impeding OA progression. The unique and complex functional demands of the two contrasting tissues that comprise osteochondral tissue require the use of bilayered scaffolds to promote individual growth of both on a single integrated implant. This paper reviews the three current bilayered scaffold strategies applied to solve this challenging problem, with a focus on the need for an innovative approach to design and fabrication of new optimized scaffold combinations to reinforce materials science as an important element of osteochondral tissue engineering.     

Simultaneous regeneration of articular cartilage and subchondral bone in vivo using MSCs induced by a spatially controlled gene delivery system in bilayered integrated scaffolds

Engineering complex tissues is important but difficult to achieve in tissue regeneration. Osteochondral tissue engineering for the repair of osteochondral defect, involving simultaneous regeneration of bone and cartilage, has attracted considerable attention and also serves as an optimal model system for developing effective strategies aimed at regenerating complex tissues. In the present study, we formulated a bilayered gene-activated osteochondral scaffold consisting of plasmid TGF-β1-activated chitosan-gelatin scaffold for chondrogenic layer and plasmid BMP-2-activated hydroxyapatite/chitosan-gelatin scaffold for osteogenic layer. Mesenchymal stem cells seeded in each layer of the bilayered gene-?activated osteochondral scaffold showed significant cell proliferation, high expression of TGF-β1 protein and BMP-2 protein respectively. The results showed that spatially controlled and localized gene delivery system in the bilayered integrated scaffolds could induce the mesenchymal stem cells in different layers to differentiate into chondrocytes and osteoblasts in vitro, respectively, and simultaneously support the articular cartilage and subchondral bone regeneration in the rabbit knee ostochondral defect model. This study gives the evidence that multi-tissue regeneration through the combination of biomimetic and multi-phasic scaffold design, spatially controlled and localized gene delivery system and multi-lineage differentiation of a single stem cell population represents a promising strategy for facilitating the development of complex tissue or organ systems.     

Dual growth factor delivery from bilayered,biodegradable hydrogel composites for spatially-guided osteochondral tissue repair

The present work investigated the use of biodegradable hydrogel composite scaffolds, based on the macromer oligo(poly(ethylene glycol) fumarate) (OPF), to deliver growth factors for the repair of osteochondral tissue in a rabbit model. In particular, bilayered OPF composites were used to mimic the structural layers of the osteochondral unit, and insulin-like growth factor-1 (IGF-1) and bone morphogenetic protein-2 (BMP-2) were loaded into gelatin microparticles and embedded within the OPF hydrogel matrix in a spatially controlled manner. Three different scaffold formulations were implanted in a medial femoral condyle osteochondral defect: 1) IGF-1 in the chondral layer, 2) BMP-2 in the subchondral layer, and 3) IGF-1 and BMP-2 in their respective separate layers. The quantity and quality of osteochondral repair was evaluated at 6 and 12 weeks with histological scoring and micro-computed tomography (micro-CT). While histological scoring results at 6 weeks showed no differences between experimental groups, micro-CT analysis revealed that the delivery of BMP-2 alone increased the number of bony trabecular islets formed, an indication of early bone formation, over that of IGF-1 delivery alone. At 12 weeks post-implantation, minimal differences were detected between the three groups for cartilage repair. However, the dual delivery of IGF-1 and BMP-2 had a higher proportion of subchondral bone repair, greater bone growth at the defect margins, and lower bone specific surface than the single delivery of IGF-1. These results suggest that the delivery of BMP-2 enhances subchondral bone formation and that, while the dual delivery of IGF-1 and BMP-2 in separate layers does not improve cartilage repair under the conditions studied, they may synergistically enhance the degree of subchondral bone formation. Overall, bilayered OPF hydrogel composites demonstrate potential as spatially-guided, multiple growth factor release vehicles for osteochondral tissue repair.     

Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral tissue-engineering applications: Scaffold design and its performance when seeded with goat bone marrow stromal cells

Recent studies suggest that bone marrow stromal cells are a potential source of osteoblasts and chondrocytes and can be used to regenerate damaged tissues using a tissue-engineering (TE) approach. However, these strategies require the use of an appropriate scaffold architecture that can support the formation de novo of either bone and cartilage tissue, or both, as in the case of osteochondral defects. The later has been attracting a great deal of attention since it is considered a difficult goal to achieve. This work consisted on developing novel hydroxyapatite/chitosan (HA/CS) bilayered scaffold by combining a sintering and a freeze-drying technique, and aims to show the potential of such type of scaffolds for being used in TE of osteochondral defects. The developed HA/CS bilayered scaffolds were characterized by Fourier transform infra-red spectroscopy, X-ray diffraction analysis, micro-computed tomography, and scanning electron microscopy (SEM). Additionally, the mechanical properties of HA/CS bilayered scaffolds were assessed under compression. In vitro tests were also carried out, in order to study the water-uptake and weight loss profile of the HA/CS bilayered scaffolds. This was done by means of soaking the scaffolds into a phosphate buffered saline for 1 up to 30 days. The intrinsic cytotoxicity of the HA scaffolds and HA/CS bilayered scaffolds extract fluids was investigated by carrying out a cellular viability assay (MTS test) using Mouse fibroblastic-like cells. Results have shown that materials do not exert any cytotoxic effect. Complementarily, in vitro (phase I) cell culture studies were carried out to evaluate the capacity of HA and CS layers to separately, support the growth and differentiation of goat marrow stromal cells (GBMCs) into osteoblasts and chondrocytes, respectively. Cell adhesion and morphology were analysed by SEM while the cell viability and proliferation were assessed by MTS test and DNA quantification. The chondrogenic differentiation of GBMCs was evaluated measuring the glucosaminoglycans synthesis. Data showed that GBMCs were able to adhere, proliferate and osteogenic differentiation was evaluated by alkaline phosphatase activity and immunocytochemistry assays after 14 days in osteogenic medium and into chondrocytes after 21 days in culture with chondrogenic medium. The obtained results concerning the physicochemical and biological properties of the developed HA/CS bilayered scaffolds, show that these constructs exhibit great potential for their use in TE strategies leading to the formation of adequate tissue substitutes for the regeneration of osteochondral defects.     

PEOT/PBT based scaffolds with low mechanical properties improve cartilage repair tissue formation in osteochondral defects

The aim of our study was to compare the healing response of biomechanically and biochemically different scaffolds in osteochondral defects created in rabbit medial femoral condyles. A block copolymer comprised of poly(ethylene oxide terephthalate) and poly(butylene terephthalate) was used to prepare porous scaffolds. The 70/30 scaffold (70 wt % poly(ethylene oxide terephthalate)) was compared to the stiffer 55/45 (55 wt % poly(ethylene oxide terephthalate)) scaffold. Nine 6-month-old rabbits were used. Osteochondral defects were filled with 55/45 scaffolds (n = 6); 70/30 scaffolds (n = 6); or left empty (n = 6). Defect sites were allowed to heal for 12 weeks. Condyles were macroscopically evaluated and analysed histologically using the O'Driscoll score for evaluating repair of osteochondral defects. Repair tissue in 70/30 scaffolds consisted of cartilage-like tissue on top of trabecular bone, whereas the tissue within the 55/45 scaffolds consisted predominantly of trabecular bone. O'Driscoll scores for 70/30 scaffolds were significantly better (p = 0.024) in comparison to untreated osteochondral defects and 55/45 scaffolds. This study reveals that the biomechanical and biochemical properties of the scaffold play an important role by themselves, and can affect the healing response of osteochondral defects. Scaffolds with low mechanical properties were superior in cartilage repair tissue formation.     

Articular chondrocytes and mesenchymal stem cells seeded on biodegradable scaffolds for the repair of cartilage in a rat osteochondral defect model

This work investigated the ability of co-cultures of articular chondrocytes and mesenchymal stem cells (MSCs) to repair articular cartilage in osteochondral defects. Bovine articular chondrocytes and rat MSCs were seeded in isolation or in co-culture onto electrospun poly(?-caprolactone) (PCL) scaffolds and implanted into an osteochondral defect in the trochlear groove of 12-week old Lewis rats. Additionally, a blank PCL scaffold and untreated defect were investigated. After 12 weeks, the extent of cartilage repair was analyzed through histological analysis, and the extent of bone healing was assessed by quantifying the total volume of mineralized bone in the defect through microcomputed tomography. Histological analysis revealed that the articular chondrocytes and co-cultures led to repair tissue that consisted of more hyaline-like cartilage tissue that was thicker and possessed more intense Safranin O staining. The MSC, blank PCL scaffold, and empty treatment groups generally led to the formation of fibrocartilage repair tissue. Microcomputed tomography revealed that while there was an equivalent amount of mineralized bone formation in the MSC, blank PCL, and empty treatment groups, the defects treated with chondrocytes or co-cultures had negligible mineralized bone formation. Overall, even with a reduced number of chondrocytes, co-cultures led to an equal level of cartilage repair compared to the chondrocyte samples, thus demonstrating the potential for the use of co-cultures of articular chondrocytes and MSCs for the in vivo repair of cartilage defects.     

Direct rAAV SOX9 administration for durable articular cartilage repair with delayed terminal differentiation and hypertrophy in vivo

Direct gene transfer strategies are of promising value to treat articular cartilage defects. Here, we tested the ability of a recombinant adeno-associated virus (rAAV) SOX9 vector to enhance the repair of cartilage lesions in vivo. The candidate construct was provided to osteochondral defects in rabbit knee joints vis-à-vis control (lacZ) vector treatment and to cells relevant of the repair tissue (mesenchymal stem cells, chondrocytes). Efficient, long-term transgene expression was noted within the lesions (up to 16 weeks) and in cells in vitro (21 days). Administration of the SOX9 vector was capable of stimulating the biological activities in vitro and over time in vivo. SOX9 treatment in vivo was well tolerated, leading to improved cartilage repair processes with enhanced production of major matrix components. Remarkably, application of rAAV SOX9 delayed premature terminal differentiation and hypertrophy in the newly formed cartilage, possible due to contrasting effects of SOX9 on RUNX2 and β-catenin osteogenic expression in this area. Most strikingly, SOX9 treatment improved the reconstitution of the subchondral bone in the defects, possibly due to an increase in RUNX2 expression in this location. These findings show the potential of direct rAAV gene delivery as an efficient tool to treat cartilage lesions.     

SOX trio-co-transduced adipose stem cells in fibrin gel to enhance cartilage repair and delay the progression of osteoarthritis in the rat

The aim of this study was to test the hypotheses that retroviral gene transfer of SOX trio enhances the in vitro chondrogenic differentiation of ASCs, and that SOX trio-co-transduced ASCs in fibrin gel promote the healing of osteochondral defects, and arrest the progression of surgically-induced osteoarthritis in a rat model. ASCs isolated from inguinal fat in rats were transduced with SOX trio genes using retrovirus, and further cultured in vitro in pellets for 21 days, then analyzed for gene and protein expression of SOX trio and chondrogenic markers. SOX trio-co-transduced ASCs in fibrin gel were implanted on the osteochondral defect created in the patellar groove of the distal femur, and also injected into the knee joints of rats with surgically-induced osteoarthritis. Rats were sacrificed after 8 weeks, and analyzed grossly and microscopically. After 21 days, ASCs transduced with SOX-5, -6, or -9 had hundreds-fold greater gene expression of each gene compared with the control with the SOX protein expression matching gene expression. SOX trio-co-transduction significantly increased GAG contents as well as type II collagen gene and protein expression. ASCs co-transduced with SOX trio significantly promoted the in vivo cartilage healing in osteochondral defect model, and prevented the progression of degenerative changes in surgically-induced osteoarthritis.     

Ectopic osteochondral formation of biomimetic porous PVA-n-HA/PA6 bilayered scaffold and BMSCs construct in rabbit

In this work, the novel poly vinyl alcohol/gelatin-nano-hydroxyapatite/polyamide6 (PVA-n-HA/PA6) bilayered scaffold with biomimetic properties for articular cartilage and subchondral bone is developed. Furthermore, when these osteochondral scaffolds were seeded with induced bone mesenchymal stem cells (BMSCs) and implanted at ectopic sites, showed the potential for an engineered cartilage tissue and the corresponding subchondral bone. BMSCs were expanded in vitro and induced to chondrogenic or osteogenic potential by culturing in suitable media for 14 days. Subsequently, these induced cells were seeded into PVA-n-HA/PA6 separately, and the constructs were implanted into the rabbit muscle pouch for upto 12 weeks. Ectopic neocartilage formation in the PVA layer and reconstitution of the subchondral bone which remained confined within the n-HA/PA6 layer with the alteration of the cellular phenotype were identified with Masson's trichrome stain. Simultaneously, the RT-PCR results confirmed the expression of specific extracellular matrix (ECM) markers for cartilaginous tissue, such as collagen type II (Col-II), or alternatively, markers for osteoid tissue, such as collagen type I (Col-I) at the corresponding layers. During ectopic implantation, the underlying subchondral bone layer was completely integrated with the cartilage layer. The result from the ectopic osteochondral scaffolds implantation suggests that PVA-n-HA/PA6 with induced BMSCs is a possible substitute with potential in cartilage repair strategies.     

羟基丁酸-羟基辛酸共聚体/胶原骨软骨一体化支架修复膝关节软骨损伤

BACKGROUND: Due to the complex physiological characteristics of the osteochondral tissue, the clinical repair of knee cartilage injury often has dissatisfied outcomes. Tissue engineering methods and tools provide a new idea for osteochondral repair. OBJECTIVE: To observe the effect of poly(hydroxybutyrate-co-hydroxyoctanoate/collagen) osteochondral tissue-engineered scaffold on the repair of articular cartilage injury in a rabbit. METHODS: The poly(hydroxybutyrate-co-hydroxyoctanoate/collagen) osteochondral tissue-engineered scaffold was prepared by solvent casting/particle leaching method. Then, seed cells were isolated and cultured on the scaffold. Twenty-four healthy New Zealand white rabbits, 4 weeks of age, were used for the study. Under balanced anesthesia, an articular cartilage defect (4.5 mm in diameter, 5 mm in depth) was created on the rabbit’s femoral condyle using a bone drill. After modeling, rabbits were randomized into three groups and given direct suture in blank group, pure scaffold implantation in control group and implantation of the scaffold-cell complex in experimental group. Femoral condyle of each rabbit was taken out for gross and histological observations at 8, 20 weeks after surgery. RESULTS AND CONCLUSION: At 8 weeks after surgery, transparent film-covered defects and small/irregular cells were found in the experimental group; the defects were filled with fibrous tissues in the control group; while there was no repair in the blank group. Until the 20th week, the defects were covered with hyaline cartilage-like tissues, accompanied by regular cell arrangement in the experimental group; in the control group, the defects were covered with white membranous tissues, and many chondrocytes were found at the basement and edge; in the blank group, some newborn tissues were visible at the defect region. These findings suggest that the poly (hydroxybutyrate-co- hydroxyoctanoate/collagen) osteochondral tissue-engineered scaffold carrying seed cells contributes to articular cartilage repair.     

Tissue engineering-based cartilage repair with allogenous chondrocytes and gelatin-chondroitin-hyaluronan tri-copolymer scaffold: a porcine model assessed at 18, 24, and 36 weeks

We previously showed that cartilage tissue can be engineered in vitro with porcine chondrocytes and gelatin/chondoitin-6-sulfate/hyaluronan tri-copolymer which mimic natural cartilage matrix for use as a scaffold. In this animal study, 15 miniature pigs were used in a randomized control study to compare tissue engineering with allogenous chondrocytes, autogenous osteochondral (OC) transplantation, and spontaneous repair for OC articular defects. In another study, 6 pigs were used as external controls in which full thickness (FT) and OC defects were either allowed to heal spontaneously or were filled with scaffold alone. After exclusion of cases with infection and secondary arthritis, the best results were obtained with autogenous OC transplantation, except that integration into host cartilage was poor. The results for the tissue engineering-treated group were satisfactory, the repair tissue being hyaline cartilage and/or fibrocartilage. Spontaneous healing and filling with scaffold alone did not result in good repair. With OC defects, the subchondral bone plate was not restored by cartilage tissue engineering. These results show that tri-copolymer can be used in in vivo cartilage tissue engineering for the treatment of FT articular defects.     

Poly-<Emphasis Type="SmallCaps">l</Emphasis>-Lactic Acid/Hydroxyapatite Electrospun Nanocomposites Induce Chondrogenic Differentiation of Human MSC

Cartilage and bone tissue engineering has been widely investigated but is still hampered by cell differentiation and transplant integration issues within the constructs. Scaffolds represent the pivotal structure of the engineered tissue and establish an environment for neo-extracellular matrix synthesis. They can be associated to signals to modulate cell activity. In this study, considering the well reported role of hydroxyapatite (HA) in cartilage repair, we focused on the putative chondrogenic differentiation of human mesenchymal stem cells (hMSCs) following culture on membranes of electrospun fibers of poly-l-lactic acid (PLLA) loaded with nanoparticles of HA. hMSCs were seeded on PLLA/HA and bare PLLA membranes and cultured in basal medium, using chondrogenic differentiation medium as a positive control. After 14 days of culture, SOX-9 positive cells could be detected in the PLLA/HA group. Cartilage specific proteoglycan immunostain confirmed the presence of neo-extracellular-matrix production. Co-expression of CD29, a typical surface marker of MSCs and SOX-9, suggested different degrees in the differentiation process. We developed a hydroxyapatite functionalized scaffold with the aim to recapitulate the native histoarchitecture and the molecular signaling of osteochondral tissue to facilitate cell differentiation toward chondrocyte. PLLA/HA nanocomposites induced differentiation of hMSCs in a chondrocyte-like phenotype with generation of a proteoglycan based matrix. This nanocomposite could be an amenable alternative scaffold for cartilage tissue engineering using hMSCs.     

Bilayered constructs aimed at osteochondral strategies: the influence of medium supplements in the osteogenic and chondrogenic differentiation of amniotic fluid-derived stem cells

The development of osteochondral tissue engineered interfaces would be a novel treatment for traumatic injuries and aging associated diseases that affect joints. This study reports the development of a bilayered scaffold, which consists of both bone and cartilage regions. On the other hand, amniotic fluid-derived stem cells (AFSCs) could be differentiated into either osteogenic or chondrogenic cells, respectively. In this study we have developed a bilayered scaffolding system, which includes a starch/polycaprolactone (SPCL) scaffold for osteogenesis and an agarose hydrogel for chondrogenesis. AFSC-seeded scaffolds were cultured for 1 or 2 weeks in an osteochondral-defined culture medium containing both osteogenic and chondrogenic differentiation factors. Additionally, the effect of the presence or absence of insulin-like growth factor-1 (IGF-1) in the culture medium was assessed. Cell viability and phenotypic expression were assessed within the constructs in order to determine the influence of the osteochondral differentiation medium. The results indicated that, after osteogenic differentiation, AFSCs that had been seeded onto SPCL scaffolds did not require osteochondral medium to maintain their phenotype, and they produced a protein-rich, mineralized extracellular matrix (ECM) for up to 2 weeks. However, AFSCs differentiated into chondrocyte-like cells appeared to require osteochondral medium, but not IGF-1, to synthesize ECM proteins and maintain the chondrogenic phenotype. Thus, although IGF-1 was not essential for creating osteochondral constructs with AFSCs in this study, the osteochondral supplements used appear to be important to generate cartilage in long-term tissue engineering approaches for osteochondral interfaces. In addition, constructs generated from agarose-SPCL bilayered scaffolds containing pre-differentiated AFSCs may be useful for potential applications in regeneration strategies for damaged or diseased joints.     

Treatment of osteochondral defects with autologous bone marrow in a hyaluronan-based delivery vehicle

The natural repair of osteochondral defects can be enhanced with biocompatible, biodegradable and bioactive materials that provide structural support and molecular cuing to stimulate repair. Since bone marrow contains osteochondral progenitor cells and bioactive agents, it is hypothesized that the combination of scaffold and bone marrow would be a superior composite material for osteochondral repair. This hypothesis will be tested by comparing the outcome of osteochondral defects filled with a fibronectin-coated hyaluronan-based sponge (ACP) with or without autologous bone marrow. Thirty-three 4-month-old rabbits received 3-mm diameter osteochondral defects that were then filled with ACP loaded or not with autologous bone marrow. Rabbits were sacrificed at 2, 3, 4, 12, and 24 weeks after surgery and the condyles processed for histologic and immunohistochemical evaluation. The defects were graded with a histologic scoring scale. Except for the 3-week specimens, the histologic appearance of the defects was similar in both groups. Four weeks after surgery, the defects were filled with bone with a top layer of cartilage well integrated with the adjacent cartilage. Twelve and 24 weeks after surgery, the defects again showed bone filling. The primary difference between the 4-week samples and the 12- and 24-week samples was that the layer of cartilage that appeared to be thinner than the adjacent cartilage. At each harvest time, the overall histologic scores of the specimens did not reveal statistical differences between the treatment groups. However, as revealed by the results of the 3-week sacrifices, bone marrow loading appeared to accelerate the first stages of the repair process. The fibronectin-coated hyaluronan-based scaffold appears to organize the natural response and facilitate the integration of the neo-cartilage with the adjacent tissue. The fundamental tissue engineering principles derived from this study should provide guidelines for the development of comparable clinical reconstructive therapies.     

Incorporation of bioactive polyvinylpyrrolidone–iodine within bilayered collagen scaffolds enhances the differentiation and subchondral osteogenesis of mesenchymal stem cells

Polyvinylpyrrolidone–iodine (Povidone-iodine, PVP-I) is widely used as an antiseptic agent for lavation during joint surgery; however, the biological effects of PVP–I on cells from joint tissue are unknown. This study examined the biocompatibility and biological effects of PVP–I on cells from joint tissue, with the aim of optimizing cell-scaffold based joint repair. Cells from joint tissue, including cartilage derived progenitor cells (CPC), subchondral bone derived osteoblast and bone marrow derived mesenchymal stem cells (BM-MSC) were isolated. The concentration-dependent effects of PVP–I on cell proliferation, migration and differentiation were evaluated. Additionally, the efficacy and mechanism of a PVP–I loaded bilayer collagen scaffold for osteochondral defect repair was investigated in a rabbit model. A micromolar concentration of PVP–I was found not to affect cell proliferation, CPC migration or extracellular matrix production. Interestingly, micromolar concentrations of PVP–I promote osteogenic differentiation of BM-MSC, as evidenced by up-regulation of RUNX2 and Osteocalcin gene expression, as well as increased mineralization on the three-dimensional scaffold. PVP–I treatment of collagen scaffolds significantly increased fibronectin binding onto the scaffold surface and collagen type I protein synthesis of cultured BM-MSC. Implantation of PVP–I treated collagen scaffolds into rabbit osteochondral defect significantly enhanced subchondral bone regeneration at 6 weeks post-surgery compared with the scaffold alone (subchondral bone histological score of 8.80 ± 1.64 vs. 3.8 ± 2.19, p < 0.05). The biocompatibility and pro-osteogenic activity of PVP–I on the cells from joint tissue and the enhanced subchondral bone formation in PVP–I treated scaffolds would thus indicate the potential of PVP–I for osteochondral defect repair.     

多孔丝素蛋白/羟基磷灰石复合脂肪间充质干细胞修复兔关节软骨及软骨下骨缺损

背景：丝素蛋白/羟基磷灰石是细胞立体培养的良好支架,是临床常用的骨缺损修复材料,具有良好的生物相容性。脂肪干细胞具有向骨及软骨细胞分化的潜能,适合骨软骨缺损修复。 目的：观察转化生长因子β1和胰岛素样生长因子1联合成软骨诱导脂肪干细胞与丝素蛋白/羟基磷灰石复合后修复兔关节软骨及软骨下骨缺损的效果。 方法：取新西兰大白兔56只,2只用于传代培养脂肪间充质干细胞,以3×109 L-1浓度接种到丝素蛋白/羟基磷灰石。其余54只新西兰大白兔,在股骨髁间制备软骨缺损模型,随机分为细胞复合材料组、单纯材料组和空白对照组,细胞复合材料组植入复合脂肪间充质干细胞的丝素蛋白/羟基磷灰石;单纯材料组植入丝素蛋白/羟基磷灰石;空白对照组不作任何植入。从大体、影像学、组织学观察比较缺损的修复情况。 结果与结论：12周时大体观察、CT、磁共振和组织学检查细胞材料复合组软骨及软骨下骨缺损区完全被软骨组织修复,修复组织与周围软骨色泽相近,支架材料基本吸收,未见明显退变和白细胞浸润,所有标本均未见丝素蛋白残留。单纯材料组缺损区缩小、部分修复,且呈纤维软骨样修复。空白对照组缺损无明显修复。提示复合脂肪间充质干细胞的丝素蛋白/羟基磷灰石修复兔关节软骨及软骨下骨缺损能力优于单纯丝素蛋白/羟基磷灰石材料。丝素蛋白/羟基磷灰石复合脂肪间充质干细胞可形成透明软骨修复动物膝关节全层软骨缺损,重建关节的解剖结构和功能,可作为新型骨软骨组织工程支架。     

Osteochondral repair using the combination of fibroblast growth factor and amorphous calcium phosphate/poly(L-lactic acid) hybrid materials

A novel amorphous calcium phosphate (ACP)/poly(L-lactic acid) (PLLA) material, which can experience morphological variations in the microstructure is supposed to be a suitable candidate as scaffold for cartilage tissue-engineering. The purpose of this study was to evaluate the efficacy of this scaffold combined with basic fibroblast growth factor (bFGF) to repair articular cartilage defects in a rabbit model. Forty-two osteochondral defects created in the femoral condyles were (a) left untreated, (b) treated by PLLA combined with bFGF, or (c) ACP/PLLA loaded with bFGF. The treatment of PLLA incorporated with bFGF improved defect filling compared with that left untreated, while the regenerated tissue was mainly fibrocartilage and showed little bone formation with only a small amount of collagen type II (Col II) and no aggrecan gene message measured. When implanted with ACP/PLLA and bFGF, most of the defects were filled with a well-established layer of cartilage tissue with abundance of cartilaginous extracellular matrix accumulation observed. Positive immunohistochemical staining of Col II was observed. High levels of Col II and aggrecan message were also detected by RT-PCR. These results indicate the feasibility of using the combination of ACP/PLLA with bFGF for cartilage repair.     

Osteochondral interface regeneration of the rabbit knee with macroscopic gradients of bioactive signals

To date, most interfacial tissue engineering approaches have used stratified designs, in which there are two or more discrete layers comprising the interface. Continuously graded interfacial designs, where there is no discrete transition from one tissue type to another, are gaining attention as an alternative to stratified designs. Given that osteochondral regeneration holds the potential to enhance cartilage regeneration by leveraging the healing capacity of the underlying bone, we endeavored to introduce a continuously-graded approach to osteochondral regeneration. The purpose of this study was thus to evaluate the performance of a novel gradient-based scaffolding approach to regenerate osteochondral defects in the New Zealand White rabbit femoral condyle. Bioactive plugs were constructed from poly(D,L-lactic-co-glycolic acid) microspheres with a continuous gradient transition between cartilage-promoting and bone-promoting growth factors. At 6 and 12 weeks of healing, results suggested that the implants provided support for the neo-synthesized tissue, and the gradient in bioactive signaling may have been beneficial for bone and cartilage regeneration compared to the blank control implant, as evidenced by histology. In addition, the effects of preseeding gradient scaffolds with umbilical cord mesenchymal stromal cells (UCMSCs) from the Wharton's jelly of New Zealand White rabbits were evaluated. Results indicated that there may be regenerative benefits to prelocalizing UCMSCs within scaffold interiors. The inclusion of bioactive factors in a gradient-based scaffolding design is a promising new treatment strategy for defect repair in the femoral condyle.     

Engineering osteochondral constructs through spatial regulation of endochondral ossification

Chondrogenically primed bone marrow-derived mesenchymal stem cells (MSCs) have been shown to become hypertrophic and undergo endochondral ossification when implanted in vivo. Modulating this endochondral phenotype may be an attractive approach to engineering the osseous phase of an osteochondral implant. The objective of this study was to engineer an osteochondral tissue by promoting endochondral ossification in one layer of a bilayered construct and stable cartilage in the other. The top half of bilayered agarose hydrogels were seeded with culture expanded chondrocytes (termed the chondral layer) and the bottom half of the bilayered agarose hydrogels with MSCs (termed the osseous layer). Constructs were cultured in chondrogenic medium for 21 days and thereafter were either maintained in chondrogenic medium, transferred to hypertrophic medium, or implanted subcutaneously into nude mice. This structured chondrogenic bilayered co-culture was found to enhance chondrogenesis in the chondral layer, appearing to help re-establish the chondrogenic phenotype that is lost in chondrocytes during monolayer expansion. Furthermore, the bilayered co-culture appeared to suppress hypertrophy and mineralization in the osseous layer. The addition of hypertrophic factors to the media was found to induce mineralization of the osseous layer in vitro. A similar result was observed in vivo where endochondral ossification was restricted to the osseous layer of the construct, leading to the development of an osteochondral tissue. This novel approach represents a potential new treatment strategy for the repair of osteochondral defects.