Repair of an articular cartilage defect using adipose-derived stem cells loaded on a polyelectrolyte complex scaffold based on poly(l-glutamic acid) and chitosan

As a synthetic polypeptide water-soluble poly(l-glutamic acid) (PLGA) was designed to fabricate scaffolds for cartilage tissue engineering. Chitosan (CHI) has been employed as a physical cross-linking component in the construction of scaffolds. PLGA/CHI scaffolds act as sponges with a swelling ratio of 760 ± 45% (mass%), showing promising biocompatibility and biodegradation. Autologous adipose-derived stem cells (ASCs) were expanded and seeded on PLGA/CHI scaffolds, ASC/scaffold constructs were then subjected to chondrogenic induction in vitro for 2 weeks. The results showed that PLGA/CHI scaffolds could effectively support ASC adherence, proliferation and chondrogenic differentiation. The ASCs/scaffold constructs were then transplanted to repair full thickness articular cartilage defects (4 mm in diameter, to the depth of subchondral bone) created in rabbit femur trochlea. Histological observations found that articular defects were covered with newly formed cartilage 6 weeks post-implantation. After 12 weeks the regenerated cartilage had integrated well with the surrounding native cartilage and subchondral bone. Toluidine blue and immunohistochemical staining confirmed similar accumulation of glycosaminoglycans and type II collagen in engineered cartilage as in native cartilage 12 weeks post-implantation. The result was further supported by quantitative analysis of extracellular matrix deposition. The compressive modulus of the engineered cartilage increased significantly from 30% of that of normal cartilage at 6 weeks to 83% at 12 weeks. Cyto-nanoindentation also showed analogous biomechanical behavior of the engineered cartilage to that of native cartilage. The results of the present study thus demonstrate the potentiality of PLGA/CHI scaffolds in cartilage tissue engineering.     

Cartilage regeneration using mesenchymal stem cells and a PLGA-gelatin/chondroitin/hyaluronate hybrid scaffold

The study was to produce a novel hybrid poly-(lactic-co-glycolic acid) (PLGA)-gelatin/chondroitin/hyaluronate (PLGA-GCH) scaffold and evaluate its potentials in cartilage repair. The porous PLGA-GCH scaffold was developed to mimic the natural extra cellular matrix of cartilage. The differentiated mesenchymal stem cells (MSCs) seeded on PLGA-GCH or PLGA scaffold were incubated in vitro and showed that, compared to PLGA scaffold, the PLGA-GCH scaffold significantly augmented the proliferation of MSCs and GAG synthesis. Then autologous differentiated MSCs/PLGA-GCH was implanted to repair full-thickness cartilage defect in rabbit, while MSCs/PLGA for the contra lateral cartilage defect (n=30). Fifteen additional rabbits without treatment for defects were used as control. Histology observation showed the MSCs/PLGA-GCH repair group had better chondrocyte morphology, integration, continuous subchondral bone, and much thicker newly formed cartilage compared with MSCs/PLGA repair group 12 and 24 weeks postoperatively. There was a significant difference in histological grading score between these two groups, which both showed much better repair than control. The present study implied that the hybrid PLGA-GCH scaffold might serve as a new way to keep the differentiation of MSCs for enhancing cartilage repair.     

Histological and biomechanical properties of regenerated articular cartilage using chondrogenic bone marrow stromal cells with a PLGA scaffold in vivo

The properties of regenerated cartilage using bone marrow-derived mesenchymal stem cells (MSCs) and poly lactic-co-glycolic acid (PLGA) scaffold composites pretreated with TGF-beta3 were investigated and compared to the non-TGF-beta3 treated MSCs/PLGA composites in a rabbit model. We prepared MSCs/PLGA scaffold composites and pretreated it with TGF-beta3 for 3 weeks prior to transplantation. Then, composites were transplanted to the osteochondral defect in the rabbit knee. After 12 weeks of transplantation, 10 of the 12 rabbits in which TGF-beta3 pretreated MSCs/PLGA scaffold composites were transplanted showed cartilaginous regeneration. In gross morphology, regenerated cartilage showed smooth, flush, and transparent features. In indentation test, this had about 80% of Young's modulus of normal articular cartilage. Histological examination demonstrated hyaline like cartilage structures with glycosaminoglycan and type II collagen expression. Histological scores were not statistically different to the normal articular cartilage. These results showed improvement of cartilage regeneration compared to the non-TGF-beta3 pretreated MSCs/PLGA scaffold composite transplanted group. Thus, we have successfully regenerated improved hyaline-like cartilage and determined the feasibility of treating damaged articular cartilage using MSCs/PLGA scaffold composite pretreated with TGF-beta3. Also, we suggest this treatment modality as another concept of cartilage tissue engineering.     

Evaluation of early-stage osteochondral defect repair using a biphasic scaffold based on a collagen-glycosaminoglycan biopolymer in a caprine model

The aim of this study was to evaluate a new collagen-GAG-calcium phosphate biphasic scaffold for the repair of surgically created osteochondral defects in goats. Comparison of morphological, histological and mechanical performance of the repair tissue was made with defects repaired using a synthetic polymer scaffold. Defects were created in the medial femoral condyle (MFC) and lateral trochlear sulcus (LTS) of Boer Cross goats and evaluated at 12 and 26 weeks. It was found that the total histology score of the collagen-GAG based biomaterial (23.8; SD 1.7) provided a significant improvement (p<0.05) over the biphasic PLGA material (19;3) and the empty control defect (17.3;1.2) in the LTS. The overall trajectory of histological and morphological improvement between 12 and 26 weeks was found to be higher for the collagen-GAG scaffold compared to the PLGA material. The occurrence of sub-chondral bone cysts was lower for the collagen-GAG scaffold with an incidence of 17% of defects, compared to 67% for the PLGA material at 26 weeks. The cartilage repair tissue for both materials evaluated was superior after 26 weeks implantation than the empty control with 75% of the collagen-GAG-treated defects showing markedly more hyaline-like cartilage and 50% of the PLGA sites exhibiting hyaline-like appearances, compared to 17% for the empty control. These early stage data indicate biphasic scaffolds based on collagen-GAG and PLGA both provide indications of satisfactory development of a structural repair to surgically prepared osteochondral defects. Furthermore, the biomaterial composition of the collagen-GAG may provide a more favourable environment for osteochondral repair.     

Evaluation of three-dimensional scaffolds prepared from poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) for growth of allogeneic chondrocytes for cartilage repair in rabbits

Articular cartilage repair using tissue engineering approach generally requires the use of an appropriate scaffold architecture that can support the formation of cartilage tissue. In this investigation, the potential of three-dimensional scaffolds made of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) was evaluated in rabbit articular cartilage defect model. Engineered PHBHHx cartilage constructs inoculated in vitro with rabbit chondrocytes for 30 days were examined. Subsequently the constructs inoculated with chondrocytes for 10 days were selected for transplantation into rabbits. After 16 weeks of in vivo implantation, both the engineered cartilage constructs and the bare scaffolds were found to be filled the defects with white cartilaginous tissue, with the engineered constructs showing histologically good subchondral bone connection and better surrounding cartilage infusion. Owing to pre-seeded chondrocytes in the PHBHHx scaffolds, better surface integrality and more accumulation of extracellular matrix (ECM) including type II collagen and sGAG were achieved in the engineered cartilage constructs. The repaired tissues possessed an average compressive modulus of 1.58MPa. For comparison, the defects without repair treatments still showed defects with fibrous tissues. These results demonstrated that PHBHHx is a useful material for cartilage tissue engineering.     

Repair of diaphyseal bone defects with calcitriol-loaded PLGA scaffolds and marrow stromal cells

Calcitriol (1,25(OH)2D3)-loaded porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds prepared by solvent casting/salt leaching method were used to repair a 1.5 cm diaphyseal segmental bone defect as a fully absorbable osteogenic biomaterial. The in vitro release of sulforhodamine B (SRB) from PLGA scaffold was measured using spectrophotometer, considering SRB as a model drug. The SRB released from SRB-incorporated PLGA scaffold during 3 months was with relatively low initial burst. The calcitriol-loaded PLGA scaffolds with or without marrow stromal cells (MSCs) were implanted in a critical-sized intercalated bone defect in rabbit femur. Defects were assessed by radiographs until 9 weeks. The bony union of the defect was observed only in the calcitriol-loaded groups. RT-PCR results indicated that MSCs, which were seeded into calcitriol-loaded scaffold, expressed an increased level of alkaline phosphatase, osteonectin, and type I collagen mRNA at day 10. After 2 and 4 weeks, the implanted scaffolds were evaluated by histology. New osteoid matrix and direct calcium deposits were more evident in calcitriol/PLGA/MSC group. Three-dimensional computed tomography and frontal tomographic images of repaired femur showed that normal femur anatomy had been restored with cortical bone with no implanted PLGA remnants at 20 weeks. It can be concluded that the porous calcitriol-loaded PLGA scaffold combined with MSCs may be a novel method for repairing the large loaded bone defect.     

Weft-knitted silk-poly(lactide-co-glycolide) mesh scaffold combined with collagen matrix and seeded with mesenchymal stem cells for rabbit Achilles tendon repair

Abstract

Natural silk fibroin fiber scaffolds have excellent mechanical properties, but degrade slowly. In this study, we used poly(lactide-co-glycolide) (PLGA, 10:90) fibers to adjust the overall degradation rate of the scaffolds and filled them with collagen to reserve space for cell growth. Silk fibroin-PLGA (36:64) mesh scaffolds were prepared using weft-knitting, filled with type I collagen, and incubated with rabbit autologous bone marrow-derived mesenchymal stem cells (MSCs). These scaffold–cells composites were implanted into rabbit Achilles tendon defects. At 16 weeks after implantation, morphological and histological observations showed formation of tendon-like tissues that expressed type I collagen mRNA and a uniformly dense distribution of collagen fibers. The maximum load of the regenerated Achilles tendon was 58.32% of normal Achilles tendon, which was significantly higher than control group without MSCs. These findings suggest that it is feasible to construct tissue engineered tendon using weft-knitted silk fibroin-PLGA fiber mesh/collagen matrix seeded with MSCs for rabbit Achilles tendon defect repair.     

仿生活性人工软骨的体外构建及其异位成软骨分化实验研究

应用先进快速成形技术(RP)制备32枚粒度均匀(尺寸均为4mm×4mm×4mm)的聚乳酸-聚羟乙酸(PLGA)人工载体,该载体经I型胶原表面修饰后均分为A、B两组。A组载体复合人骨形态发生蛋白-2基因转染(rAAV-hBM P-2)的兔骨髓基质细胞(M SC s,2×104个细胞/枚);B组每枚载体复合等量、同代次、未基因转染M SC s。体外培养第5 d,从两组各取12枚细胞-载体复合物植入裸鼠皮下,术后30 d取材观察。结果发现rAAV-hBM P-2转染的M SC s成功表达目的基因。RP制备的PLGA载体具有良好的空间结构,大孔及材料表面微孔孔径分别为300μm和3~5μm。体外培养3~5 d,两组载体均复合生长着大量种子细胞。皮下埋植30 d,A组植入物形成较为典型的软骨细胞及基质,II型胶原蛋白表达阳性;同期B组植入物无软骨组织形成。A组聚酯材料面积百分率显著低于B组(P<0.01)。结果表明RP结合载体材料表面修饰,能制备出兼具理想孔隙结构和良好生物相容性的组织工程支架载体,该载体高效复合rAAV-hBM P-2转染的M SC s为组织工程软骨构建创造有利条件。     

丝素蛋白-壳聚糖支架复合骨髓间充质干细胞体内构建组织工程化软骨的生物相容性

文题释义： 生物相容性：是指生命体组织对非活性材料产生的一种性能,一般是指材料与宿主之间的相容性,包括组织相容性和血液相容性。 检测相容性的方法：是将支架材料与种子细胞在体外共培养,检测支架毒性、细胞活性、细胞增殖及细胞与支架的黏附情况等指标,该方法具有客观性强、可重复性强、影响因素相对简单及敏感性高等特点。 背景：课题组前期的研究中发现,丝素蛋白-壳聚糖支架材料复合诱导后骨髓间充质干细胞在兔体内能修复缺损的软骨组织,但对于该组织工程化软骨组织的生物相容性还未进一步研究。 目的：研究丝素蛋白-壳聚糖支架材料复合骨髓间充质干细胞在体内构建组织工程化软骨的生物相容性。 方法：使用丝素蛋白-壳聚糖按1∶1比例混合制备三维支架材料,提取兔骨髓间充质干细胞,将诱导后的骨髓间充质干细胞与丝素蛋白-壳聚糖支架构建修复体,再将修复体移植到兔关节软骨缺损模型中修复软骨组织。实验分为3组,实验组植入诱导后骨髓间充质干细胞+丝素蛋白-壳聚糖支架,对照组植入丝素蛋白-壳聚糖支架干预,空白组未植入修复体。 结果与结论：①实验成功制备丝素蛋白-壳聚糖三维支架材料及提取骨髓间充质干细胞,并构建软骨缺损的修复体,将修复体植入兔体内能成功修复缺损的软骨组织;②建模后2,4,8,12周,3组血常规、降钙素原、血沉、C-反应蛋白结果提示无明显的全身感染征象,3组血常规及肝肾功能各时间段比较差异无显著性意义(P > 0.05);③一般观察、苏木精-伊红染色及扫描电镜观察：建模后12周,相比其他两组,实验组软骨缺损已修复,支架材料已吸收,修复组织周围未见炎性细胞,修复组织已正常组织整合良好;④结果证实,丝素蛋白-壳聚糖支架复合骨髓间充质干细胞在体内构建的组织工程化软骨具有良好的生物相容性。 ORCID: 0000-0002-8139-1175(佘荣峰) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

The bone formation in vitro and mandibular defect repair using PLGA porous scaffolds

Highly porous scaffolds of poly(lactide-co-glycolide) (PLGA) were prepared by solution-casting/salt-leaching method. The in vitro degradation behavior of PLGA scaffold was investigated by measuring the change of normalized weight, water absorption, pH, and molecular weight during degradation period. Mesenchymal stem cells (MSCs) were seeded and cultured in three-dimensional PLGA scaffolds to fabricate in vitro tissue engineering bone, which was investigated by cell morphology, cell number and deposition of mineralized matrix. The proliferation of seeded MSCs and their differentiated function were demonstrated by experimental results. To compare the reconstructive functions of different groups, mandibular defect repair of rabbit was made with PLGA/MSCs tissue engineering bone, control PLGA scaffold, and blank group without scaffold. Histopathologic methods were used to estimate the reconstructive functions. The result suggests that it is feasible to regenerate bone tissue in vitro using PLGA foams with pore size ranging from 100-250 microm as scaffolding for the transplantation of MSCs, and the PLGA/MSCs tissue engineering bone can greatly promote cell growth and have better healing functions for mandibular defect repair. The defect can be completely recuperated after 3 months with PLGA/MSCs tissue engineering bone, and the contrastive experiments show that the defects could not be repaired with blank PLGA scaffold. PLGA/MSCs tissue engineering bone has great potential as appropriate replacement for successful repair of bone defect.     

Mesenchymal stem cell-based repair of articular cartilage with polyglycolic acid-hydroxyapatite biphasic scaffold

This study investigates the capacity of a composite scaffold composed of polyglycolic acid-hydroxyapatite (PGA-HA) and autologous mesenchymal stem cells (MSCs) to promote repair of osteochondral defects. MSCs from culture-expanded rabbits were seeded onto a PGA and HA scaffold. After a 72-hour co-culture period, the cell-adhered PGA and HA were joined together, forming an MSCs-PGA-HA composite. Full-thickness cartilage defects in the intercondylar fossa of the femur were then implanted with the MSC-PGA-HA composite, the PGA-HA scaffold only, or they were left empty (n=20). Animals were sacrificed 16 or 32 weeks after surgery and the gross appearance of the defects was evaluated. The specimens were examined histologically for morphologic features, and stained immunohistochemically for type 2 collagen. Specimens of the MSCs-PGA-HA composite implantation group demonstrated hyaline cartilage and a complete subchondral bone formation. At 16 weeks post-implantation, significant integration of the newly formed tissue with surrounding normal cartilage and subchondral bone was observed when compared to the two control groups. At 32 weeks, no sign of progressive degeneration of the newly formed tissue was found. A significant difference in histological grading score was found compared with the control groups. The novel MSCs-seeded, PGA-HA biphasic graft facilitated both articular cartilage and subchondral bone regeneration in an animal model and might serve as a new approach for clinical applications.     

Injecting partially digested cartilage fragments into a biphasic scaffold to generate osteochondral composites in a nude mice model

This study proposed a novel scaffold with heterogeneous morphology that mimics the natural tissue. Its upper part contains a hollow cavity surrounded by a wall of poly(L-lactic-co-glycolic acid) (PLGA) porous membrane for injecting cartilage tissue and cells. An interconnecting porous structure located under the hollow cavity was made of composite materials that combined PLGA and beta-tricalcium phosphate (beta-TCP) to simulate the subchondral bone. Adult pig articular cartilage was cut and sieved into small fragments. The tissue fragments was partially digested by 0.1% collagenase for 0, 2, 4, and 6 h and injected into the hollow cavity of the biphasic scaffold. The biphasic scaffolds were then implanted into the subcutaneous pocket of nude mice for 4 weeks. No tissue bonding or new cartilaginous tissue formation was identified in the cartilage fragment without enzymatic treatment. The cartilage fragments digested with 2 h of collagenase digestion were partially integrated after implantation. The integrative properties of the cartilage fragment depended on the extent of enzymatic digestion. Releasing cells at the tissue surface enhanced confluence and bonding of the cartilage fragment matrix. Complete integration of the cartilage fragments and cartilage remodeling were achieved by digestion of the tissue fragments with 4 h of enzymatic treatment. The neocartilage grew from the upper hollow cavity into the lower PLGA/beta-TCP porous structure, forming an interface similar to that formed between cartilage and subchondral bone. This study combined the osteochondral scaffold and limited cartilage tissues to generate cartilage tissue in vivo intending for repairing full-thickness articular cartilage defects.     

In vitro cartilage tissue engineering with 3D porous aqueous-derived silk scaffolds and mesenchymal stem cells

Adult cartilage tissue has limited self-repair capacity, especially in the case of severe damages caused by developmental abnormalities, trauma, or aging-related degeneration like osteoarthritis. Adult mesenchymal stem cells (MSCs) have the potential to differentiate into cells of different lineages including bone, cartilage, and fat. In vitro cartilage tissue engineering using autologous MSCs and three-dimensional (3-D) porous scaffolds has the potential for the successful repair of severe cartilage damage. Ideally, scaffolds designed for cartilage tissue engineering should have optimal structural and mechanical properties, excellent biocompatibility, controlled degradation rate, and good handling characteristics. In the present work, a novel, highly porous silk scaffold was developed by an aqueous process according to these criteria and subsequently combined with MSCs for in vitro cartilage tissue engineering. Chondrogenesis of MSCs in the silk scaffold was evident by real-time RT-PCR analysis for cartilage-specific ECM gene markers, histological and immunohistochemical evaluations of cartilage-specific ECM components. Dexamethasone and TGF-beta3 were essential for the survival, proliferation and chondrogenesis of MSCs in the silk scaffolds. The attachment, proliferation, and differentiation of MSCs in the silk scaffold showed unique characteristics. After 3 weeks of cultivation, the spatial cell arrangement and the collagen type-II distribution in the MSCs-silk scaffold constructs resembles those in native articular cartilage tissue, suggesting promise for these novel 3-D degradable silk-based scaffolds in MSC-based cartilage repair. Further in vivo evaluation is necessary to fully recognize the clinical relevance of these observations.     

Development of mature cartilage constructs using novel three-dimensional porous scaffolds for enhanced repair of osteochondral defects

In this study, we successfully developed two types of volume-reduced three-dimensional scaffolds, including cushion- and cylinder-shape scaffolds, fabricated from chitosan-based hyaluronic acid hybrid polymer fibers. Using these scaffolds combined with a bioreactor system, we regenerated histologically and mechanically mature cartilage constructs. The final goal of this study was to clarify the ability of this engineered cartilage construct to induce cartilage repair in osteochondral defects. The mature cartilage constructs regenerated with two types of scaffolds were implanted into 5-mm diameter osteochondral defects in the patellar groove of rabbits. At 12 weeks after implantation, the reparative tissues consisted of hyaline-like cartilage with evidence of stable fusion to adjacent native cartilage and normal reconstitution of subchondral bone. The histological score of these tissues significantly outranked the value of untreated tissue. Biomechanically, compression modulus of reparative tissue at 12 weeks postoperatively was comparative to that of normal articular cartilage. Our results indicate that the implantation of constructs with mature cartilage have potential as a better approach for joint resurfacing.     

Bone augmentation by bone marrow mesenchymal stem cells cultured in three-dimensional biodegradable polymer scaffolds

Poly-lactic-glycolic acid (PLGA) is a biocompatible as well as biodegradable polymer and used in various medical applications. In this study, we evaluated efficiency of the specially designed three-dimensional porous PLGA as a scaffold for bone augmentation. First, cell attachment/proliferation, differentiation, and mineralization of Fisher 344 rat marrow mesenchymal stem cells (MSCs) cultured on the PLGA scaffold were analyzed. Viable MSCs were impregnated into pore areas of the scaffold and a moderate increase of DNA contents was seen. High alkaline phosphatase, osteocalcin content, and calcium content of MSCs in PLGA scaffolds under osteogenic differentiation conditions were seen after 14 or 21 days of culture. Subsequently, we implanted the PLGA/MSCs composites on rat calvaria bone for 30 days. Newly formed bone was seen in only the composite PLGA/MSCs implantation group, which had been precultured under osteogenic condition. We also demonstrated that the newly formed bone originated from the donor composites. These results demonstrate that the three-dimensional PLGA scaffold can support osteogenic differentiation of MSCs, and the scaffold combined with osteogenic MSCs can be used for in vivo bone tissue augmentation.     

丝素蛋白/羟基磷灰石材料复合骨髓间充质干细胞构建组织工程化软骨

背景：随着组织工程的兴起,软骨损伤的修复可能性显著地提高,但单一的支架材料均不能符合理想支架,有一定的局限性。 目的：观察骨髓间充质干细胞复合丝素蛋白/羟基磷灰石构建组织工程化软骨的可行性。 方法：体外分离培养骨髓间充质干细胞,并定向诱导成软骨细胞,与丝素蛋白/羟基磷灰石复合培养,构建膝关节胫骨平台全层关节软骨缺损。54只大白兔单侧膝关节全层软骨缺损模型后随机抽签法分为3组,复合组植入细胞-丝素蛋白/羟基磷灰石复合物;材料组植入单纯丝素蛋白/羟基磷灰石,对照组不行任何植入。植入后8,12周CT检查及组织学检查观察软骨缺损修复情况。 结果与结论：植入后8周,复合组关节面不平整,关节间隙增大,形成新生类软骨细胞,基质丰富。材料组关节面塌陷,软骨细胞少量增殖。植入后12周,复合组关节面平整,关节间隙如常。大量软骨细胞出现,与周边软骨色泽一样,支架材料完全降解。材料组关节面不平整,软骨细胞不完全充填,支架材料部分降解。对照组未见修复。提示用骨髓间充质干细胞复合丝素蛋白/羟基磷灰石可形成透明软骨修复动物膝关节全层软骨缺损,显示了丝素蛋白/羟基磷灰石材料作为关节软骨组织工程支架材料的良好生物相容性。     

Porous gelatin-chondroitin-hyaluronate tri-copolymer scaffold containing microspheres loaded with TGF-beta1 induces differentiation of mesenchymal stem cells in vivo for enhancing cartilage repair

The aim of the study was to produce a novel porous gelatin-chondroitin-hyaluronate scaffold in combination with a controlled release of transforming growth factor beta1 (TGF-beta1), which induced the differentiation of mesenchymal stem cells (MSCs) in vivo for enhancing cartilage repair. Gelatin microspheres loaded with TGF-beta1 (MS-TGFbeta1) showed a fast release at the initial phase (37.4%), and the ultimate accumulated release was 83.1% by day 18. The autologous MSCs seeded on MS-TGFbeta1/scaffold were implanted to repair full-thickness cartilage defects in rabbits as in vivo differentiation repair group, while MSCs differentiated in vitro were seeded on scaffold without MS-TGFbeta1 to repair the contra lateral cartilage defects (n = 30). Fifteen additional rabbits without treatment for defects were used as control. Histology observation showed that the in vivo differentiation repair group had better chondrocyte morphology, integration, continuous subchondral bone, and much thicker newly formed cartilage layer when compared to in vitro differentiation repair group 12 and 24 weeks, postoperatively. There was a significant difference in histological grading score between these two experimental groups, and both showed much better repair than that of the control. The present study implied that the novel scaffold with MS-TGFbeta1 might serve as a new way to induce the differentiation of MSCs in vivo to enhance the cartilage repair.     

Chondrogenesis from human placenta-derived mesenchymal stem cells in three-dimensional scaffolds for cartilage tissue engineering

Human placenta-derived mesenchymal stem cells (hPMSCs) represent a promising source of stem cells. The application of hPMSCs in cartilage tissue engineering, however, was less reported. In this study, hPMSCs were grown in a three-dimensional (3D) environment for cartilage tissue formation in vitro. To select proper scaffolds for 3D culture of mesenchymal stem cells (MSCs), rat adipose-derived MSCs were initially employed to optimize the composition and condition of the 3D environment. The suitability of a poly(D,L-lactide-co-glycolide) (PLGA) precision scaffold previously developed for seeding and culture of primary chondrocytes was tested for MSCs. It was established that MSCs had to be embedded in alginate gel before seeded in the PLGA precision scaffold for cartilage-like tissue formation. The inclusion of nano-sized calcium-deficient hydroxyapatite (nCDHA) and/or a recombinant protein containing arginine-glycine-aspartate (RGD) into the alginate gel enhanced the chondrogenesis for both rat adipose-derived MSCs and hPMSCs. The amount of extracellular matrix such as glycosaminoglycan and type II collagen accumulated during a period of 21 days was found to be the greatest for hPMSCs embedded in the alginate/nCDHA/RGD gel and injected and cultivated in the precision scaffold. Also, histological analyses revealed the lacunae formation and extracellular matrix production from the seeded hPMSCs. Comparing human bone marrow-derived MSCs (hBMSCs) and hPMSCs grown in the previous composite scaffolds, the secretion of glycosaminoglycan was twice as higher for hPMSCs as that for hBMSCs. It was concluded that the alginate/nCDHA/RGD mixed gel in the aforementioned system could provide a 3D environment for the chondrogenesis of hPMSCs, and the PLGA precision scaffold could provide the dimensional stability of the whole construct. This study also suggested that hPMSCs, when grown in a suitable scaffold, may be a good source of stem cells for building up the tissue-engineered cartilage.     

Cartilage tissue engineering on the surface of a novel gelatin-calcium-phosphate biphasic scaffold in a double-chamber bioreactor

Tissue engineering is a new approach to articular cartilage repair; however, the integration of the engineered cartilage into the host subchondral bone is a major problem in osteochondral injury. The aim of the present work, therefore, was to make a tissue-engineered osteochondral construct from a novel biphasic scaffold in a newly designed double-chamber bioreactor. This bioreactor was designed to coculture chondrocytes and osteoblasts simultaneously. The aim of this study was to prove that engineered cartilage could be formed with the use of this biphasic scaffold. The scaffold was constructed from gelatin and a calcium-phosphate block made from calcined bovine bone. The cartilage part of the scaffold had a uniform pore size of about 180 microm and approximate porosity of 75%, with the trabecular pattern preserved in the bony part of the scaffold. The biphasic scaffolds were seeded with porcine chondrocytes and cultured in a double-chamber bioreactor for 2 or 4 weeks. The chondrocytes were homogeneously distributed in the gelatin part of the scaffold, and secretion of the extracellular matrix was demonstrated histologically. The chondrocytes retained their phenotype after 4 weeks of culture, as proven immunohistochemically. After 4 weeks of culture, hyaline-like cartilage with lacuna formation could be clearly seen in the gelatin scaffold on the surface of the calcium phosphate. The results show that this biphasic scaffold can support cartilage formation on a calcium-phosphate surface in a double-chamber bioreactor, and it seems reasonable to suggest that there is potential for further application in osteochondral tissue engineering.     

In vivo degradation of porous poly(propylene fumarate)/poly(DL-lactic-co-glycolic acid) composite scaffolds

This study investigated the in vivo degradation of poly(propylene fumarate) (PPF)/poly(DL-lactic-co-glycolic acid) (PLGA) composite scaffolds designed for controlled release of osteogenic factors. PPF/PLGA composites were implanted into 15.0mm segmental defects in the rabbit radius, harvested after 12 and 18 weeks, and analyzed using histological techniques to assess the extent of polymer degradation as well as the tissue response within the pores of the scaffolds. Polymer degradation was limited to micro-fragmentation of the scaffold at the ends and edges of the implant at both 12 and 18 weeks. The tissue within the pores of the scaffold consisted of fibrous tissue, blood vessels and some inflammatory cells. In areas where polymer breakdown was evident, an increased inflammatory response was observed. In contrast, areas of bone ingrowth into the polymer scaffold were characterized by minimal inflammatory response and polymer degradation. Our results show that minimal degradation of porous PPF occurs within 18 weeks of implantation in a rabbit model. Further, the in vivo degradation data of porous PPF/PLGA scaffolds are comparable with earlier obtained in vitro data.