Rotating three‐dimensional dynamic culture of adult human bone marrow‐derived cells for tissue engineering of hyaline cartilage

The method of constructing cartilage tissue from bone marrow‐derived cells in vitro is considered a valuable technique for hyaline cartilage regenerative medicine. Using a rotating wall vessel (RWV) bioreactor developed in a NASA space experiment, we attempted to efficiently construct hyaline cartilage tissue from human bone marrow‐derived cells without using a scaffold. Bone marrow aspirates were obtained from the iliac crest of nine patients during orthopedic operation. After their proliferation in monolayer culture, the adherent cells were cultured in the RWV bioreactor with chondrogenic medium for 2 weeks. Cells from the same source were cultured in pellet culture as controls. Histological and immunohistological evaluations (collagen type I and II) and quantification of glycosaminoglycan were performed on formed tissues and compared. The engineered constructs obtained using the RWV bioreactor showed strong features of hyaline cartilage in terms of their morphology as determined by histological and immunohistological evaluations. The glycosaminoglycan contents per µg DNA of the tissues were 10.01 ± 3.49 µg/µg DNA in the case of the RWV bioreactor and 6.27 ± 3.41 µg/µg DNA in the case of the pellet culture, and their difference was significant. The RWV bioreactor could provide an excellent environment for three‐dimensional cartilage tissue architecture that can promote the chondrogenic differentiation of adult human bone marrow‐derived cells. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 517–521, 2009     

成软骨诱导的骨髓间充质干细胞膜片修复肋软骨供区缺损的实验研究

目的采用兔胸廓损伤动物模型,观察成软骨诱导的骨髓间充质干细胞膜片对肋软骨供区再生修复的影响。方法将16只家兔随机分为4组,每组4只,分别为健康对照组,实验1、2、3组。健康对照组家兔无任何处理,对实验组的每组双侧第4—6肋软骨均采用不同的2种方法处理,同侧3根肋软骨采用同一种方法处理,3种方法在每组中两两配对。3种方法分别为：①直接缝合软骨膜;②骨髓间充质干细胞膜片折叠数层成圆筒状填塞人肋软骨缺损处缝合;③成软骨诱导的骨髓间充质干细胞膜片同法折叠数层成圆筒状填塞入肋软骨缺损处,缝合封闭缺损。3种方法在各实验组兔两侧肋软骨中两两配对,健康对照组不做处理。术后16周,处死家兔取材进行大体观察,常规HE染色,并行生物力学检测,测定所有肋软骨的抗压强度及弯曲强度。结果各实验组家兔的胸廓整体形态均较良好,各组及各处理方法间无明显差别。生物力学检测显示,3种处理方法之间均存在差异（P〈0．01）,方法3处理的修复组织的抗压、弯曲强度与健康对照组比较,差异无统计学意义（P〉0．05）;方法1、2处理的修复组织的抗压、弯曲强度明显低于健康对照组（P〈0．01）;方法2处理的修复组织的抗压、弯曲强度优于方法1。组织切片HE染色病理观察,可见方法1、2处理的修复组织主要为纤维组织,方法3处理的修复组织内,可见新生的软骨细胞和大量的软骨细胞外基质。结论成软骨诱导的骨髓间充质干细胞膜片可以促进肋软骨供区软骨细胞的再生,修复肋软骨供区缺损,维持胸廓的正常形态和稳定性,从而降低术后胸廓畸形的发生率。     

人真皮成纤维细胞体外构建组织工程化软骨的初步探索

目的 利用软骨细胞提供的体外软骨诱导微环境,探讨人真皮成纤维细胞在体外构建软骨的可行性.方法 分别培养猪的软骨细胞与人真皮成纤维细胞,将2种细胞按1:1(软骨细胞:成纤维细胞)比例混匀,以5.0×10~7/ml的终浓度接种于聚羟基乙酸支架(PGA,直径9 mm,高2mm)作为共培养组,相同终浓度的单纯软骨细胞和单纯成纤维细胞分别接种于相同支架作为阳性对照及阴性对照.每组各接种3个标本,每个接种细胞悬液200 μl.全部标本均于体外培养8周后取材,通过大体观察、湿重测定、组织学及免疫组化等相关检测对构建软骨进行评价.结果 软骨细胞组(阳性对照组)基本保持了复合物初始的大小和形状,组织周边和中央均有较均质的软骨陷窝样结构形成,表达软骨特异性细胞外基质;共培养组的组织稍有缩小,组织周边也有软骨陷窝样结构形成,表达软骨特异性细胞外基质,但组织内大部分区域形成了纤维样组织,特别是通过人核抗原免疫组化和对应的Safranin O染色结果,可以看到少量人核抗原阳性的细胞形成了较成熟的陷窝样结构,表达软骨特异性基质.单纯成纤维细胞组(阴性对照组)在体外培养过程中逐渐皱缩变形,未形成软骨样组织.结论 软骨细胞共培养体系可以有效地诱导人真皮成纤维细胞中一定比例的细胞向软骨分化,并能在体外构建软骨样组织.     

The Cartilaginous Potential of the Perichondrium in Rabbit Ear and Rib

In an experimental study the cartilaginous potential of the rabbit ear perichondrium has been compared with that of the rib in vivo and in vitro. Perichondrium was transferred as free autologous grafts to the subcutaneous tissue on the scalp and as loose bodies into the knee joint. The presence of cartilage in the grafts was examined after six weeks. In vitro explants of rabbit perichondrium from the ear and the rib were maintained in an organ culture system. The presence of cartilage was analyzed after one to three weeks. Rabbit perichondrium from the rib appeared to have a greater cartilaginous potential than that from the ear both in vivo and in vitro. Chondrogenesis in perichondrium was demonstrated in vitro.     

The potential of adult human perichondrium to form hyalin cartilage in vitro

The usefulness of adult human perichondrium for the restoration of articular cartilage defects depends on the potential to form hyalin cartilage. In order to evaluate the capacity of adult human perichondrium to form hyalin cartilage in vitro, perichondrium of the rib of eight adult human beings was cultured in vitro. After removal of residual cartilage, perichondrial explants were cultured for 7 or 10 days. The explants were histologically examined using specific stains to prove the presence of glycosaminoglycans (GAGs) normal for hyalin cartilage. Clear differentiation of perichondrial cells towards chondrocytes was noted. The chondrocytes synthesized new matrix substances normally present in hyalin cartilage. This investigation supports the usefulness of adult human rib perichondrium for the restoration of cartilage defects. Due to the enormous potential of the rib perichondrium to form hyalin cartilage in vitro, even defects in joints with a rather thick cartilage layer might be restored using this biological material.     

Characterisation of cells in regenerating cartilage from autotransplanted perichondrium. immunohistochemical expression of smooth-muscle actin, desmin, vimentin, and Ki-67.

Autotransplanted perichondrium from rib and ear sutured to the knee joints of 26 rabbits has been examined with immunohistochemistry and shows certain structural, functional, and proliferative characteristics of regenerating cartilage. Cryostat sections have been examined for the expression of smooth-muscle actin (SMA), desmin, vimentin, and Ki-67. In this rabbit model of perichondrial grafting SMA staining showed vivid vessel regeneration, particularly in the proliferating stage about two to three weeks after grafting, and no vessels in more mature parts one month or more after transplantation. Desmin staining showed expression and distribution similar to SMA. Vimentin staining shows the cytoskeleton of regenerating cartilaginous tissue and makes cellular borders apparent. Immunohistochemical expression of Ki-67 is constantly negative in perichondrial tissue from rib and ear before transplantation, clearly positive in the proliferative stage, but there is no expression in maturing cartilage. The study also shows that all human antibodies used are applicable in a rabbit model.     

骨髓间充质干细胞体外构建组织工程化半月板

目的探索以骨髓间充质干细胞（BMSCs）为种子细胞,在体外构建具有完整内侧半月板形态的软骨样组织的方法。方法运用模具制备内侧半月板形的PGA/PLA支架。抽取犬骨髓,分离培养BMSCs,将其接种于支架材料上,5 d后使用软骨诱导液培养。体外培养6周后,行大体观察、组织学检测及生物力学检测。结果细胞材料复合物能够较好地维持半月板三维立体结构,形成了表面光滑、触之有弹性的瓷白色软骨样组织。 HE染色可见典型的软骨陷窝出现,说明成熟软骨组织的形成。Safranine O染色证实有蛋白聚糖基质产生。生物力学检测显示,新生组织弹性模量达正常半月板组织的12.7%。结论 BMSCs通过体外诱导,可在体外分化为较成熟的软骨组织,并构建出组织工程化半月板。     

Ectopic mineralized cartilage formation in human undifferentiated pancreatic adenocarcinoma explants grown in nude mice

Mineralized as well as nonmineralized cartilage-like structures enclosing cells resembling chondrocytes were found in human-derived undifferentiated but not in poorly differentiated pancreatic adenocarcinoma explants grown in nude mice. The structures reacted with anti-mouse IgG but not with antibodies against human cytokeratin 19, indicating that the newly formed tissue was of mouse origin. High activity of alkaline phosphatase was found in cell layers surrounding the structures and in cells embedded in the matrix. The extracellular matrix was strongly positive after Sirius red staining, reacted with anti-collagen type II antibodies, and the presence of proteoglycans was demonstrated with Alcian blue staining and by metachromasia after Giemsa staining. Electron microscopic inspection revealed the presence of bundles of both thick collagenous fibrils with low levels of fine filamentous material and thin collagenous fibrils with high concentrations of filamentous components. The majority of both types of matrices was found to be partially or completely calcified. The mean area density of the cartilage-like structures in the undifferentiated tumors was 0.31%. The frequent formation of the cartilage-like structures in the rapidly growing undifferentiated explants and its absence in the slowly growing, more differentiated explants suggest that low oxygen tensions in combination with altered levels of growth factors, such as members of the transforming growth factor superfamily, create conditions that induce differentiation of fibroblasts to chondrocytes. It is concluded that these human tumors grown in nude mice can be used as an in vivo model to study ectopic formation of mineralized cartilage.     

Repair of large osteochondral defects with allogeneic cartilaginous aggregates formed from bone marrow-derived cells using RWV bioreactor.

Our objective was to examine the technique of regenerating cartilage tissue from bone marrow-derived cells by three-dimensional (3D) culture using the rotating wall vessel (RWV) bioreactor. Three-dimensional and cylindrical aggregates of allogeneic cartilage with dimensions of 10 x 5 mm (height x diameter) formed by the RWV bioreactor were transplanted into osteochondral defects of Japanese white rabbits (Group T, n = 15). For the control, some osteochondral defects were left empty (Group C, n = 18). At 4, 8, and 12 weeks postimplantation, the reparative tissues were evaluated macroscopically, histologically, and biochemically. In Group T at as early as 4 weeks, histological observation, especially via safranin-O staining, suggested that the reparative tissues resembled hyaline cartilage. And we observed no fibrous tissues between reparative tissue and adjacent normal tissues. In the deeper portion of the bony compartment, the osseous tissues were well remodeled. At 4 and 8 weeks postimplantation, the mean histological score of Group T was significantly better than that of Group C (p < 0.05). The glycosaminoglycans (GAG)/DNA ratio in both groups increased gradually from 4 to 8 weeks and then decreased from 8 to 12 weeks. We herein report the first successful regeneration of cartilage in osteochondral defects in vivo using allogeneic cartilaginous aggregates derived from bone marrow-derived cells by 3D culture using the RWV bioreactor.     

The influence of human amniotic fluid on the potential of rabbit ear perichondrial flaps to form cartilage tissue.

Since several experimental and clinical studies demonstrated the chondrogenic potential of perichondrium, there has been great interest in examining factors that might promote neochondrogenesis from perichondrium. Human amniotic fluid contains hyaluronic acid, growth factors and extracellular macromolecules, and may, therefore, have a stimulating effect on cartilage regeneration. This experimental study investigated the effect of human amniotic fluid on cartilage regeneration from rabbit ear perichondrial flaps, using 96 ears of 48 New Zealand young rabbits. A perichondrial flap was elevated and a cartilage defect measuring 20 mm x 15 mm was created on the dorsum of each ear, then the perichondrial flap was sutured in place. The ears were divided into two groups according to the solution injected underneath the perichondrial flap. The right ears, which were injected with 0.2 ml human amniotic fluid, formed the experimental group, and the left ears, which were injected with 0.2 ml saline, formed the control group. Macroscopic and histological progression of neochondrogenesis were evaluated at 2, 4, 6 and 8 weeks after surgery. Macroscopically, the cartilage in the experimental group was generated quickly and had a similar appearance to the surrounding cartilage tissue, whereas in the control group minimal cartilage formation was observed at 4 weeks. Histologically, the neocartilage was significantly thicker in the experimental group than in the control group at 8 weeks (P < 0.05, Student's t -test). It can be concluded that human amniotic fluid enhances new cartilage formation from rabbit ear perichondrial flaps. The preventive effect of human amniotic fluid on scar formation and the rich content of growth factors and extracellular matrix precursors may play a role in this result.     

In vitro engineering of human autogenous cartilage.

A challenge in tissue engineering is the in vitro generation of human cartilage. To meet standards for in vitro-engineered cartilage, such as prevention of immune response and structural as well as functional integration to surrounding tissue, we established a three-dimensional cell culture system without adding exogenous growth factors or scaffolds. Human chondrocytes were cultured as spheroids. Tissue morphology and protein expression was analyzed using histological and immunohistochemical investigations on spheroid cryosections. A cartilage-like tissue similar to naturally occurring cartilage was generated when spheroids were cultured in medium supplemented only with human serum. This in vitro tissue was characterized by the synthesis of the hyaline-specific proteins collagen type II and S-100, as well as the synthesis of hyaline-specific mucopolysaccharides that increased with prolonged culture time. After 3 months, cell number in the interior of in vitro tissues was diminished and was only twice as much as in native cartilage. Additionally, spheroids quickly adhered to and migrated on glass slides and on human condyle cartilage. The addition of antibiotics to autologous spheroid cultures inhibited the synthesis of matrix proteins. Remarkably, replacing human serum by fetal calf serum resulted in the destruction of the inner part of the spheroids and only a viable rim of cells remained on the surface. These results show that the spheroid culture allows for the first time the autogenous in vitro engineering of human cartilage-like tissue where medium supplements were restricted to human serum.     

Tissue engineered cartilage generated from human trachea using DegraPol scaffold.

OBJECTIVE: To date numerous attempts have been undertaken to conquer the challenging problem of reconstructing long segmental tracheal defects, as yet without lasting success. Recently, employing concepts of tissue engineering in animals, cartilage-like constructs were transplanted in vivo. However, both the feasibility of fabricating tracheal replacements and the use of human tracheal chondrocytes (HTC) for tissue engineering are still under investigation. In this study, we optimized isolation and cultivation techniques for human tracheal cartilage, assessing the feasibility of seeding these cells onto a novel, three-dimensional (3-D) polyester-urethane polymer (DegraPol). METHODS: Human tracheal cartilage was harvested from the trachea of lung donors, digested in 0.3% collagenase II, and the condrocytes serially passaged every 7-9 days. Cells were also cultivated over agar plate during the total 6-8 weeks expansion phase. Thereafter, chondrocytes were seeded onto DegraPol (pore sizes 150-200 microm) with a seeding density of 2.4 x 10(7)/ml, and chondrocyte-polymer constructs maintained during in vitro static culture. RESULTS: HTC displayed stable proliferation kinetics in monolayer culture with positive expression of collagen type II. Following polymer seeding, both cellular proliferation and extracellular matrix (ECM) production, as measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and glycosaminoglycan assays, continued over extended culture. Active growth of HTC on DegraPol was further demonstrated by Alcian blue staining, with the histomorphological appearance of the construct resembling that of native cartilage. Scanning electron microscopy showed chondrocyte growth and ECM synthesis both on the surface and inside the porous scaffold, with a dense cell layer on the surface of the scaffold and a lower cell distribution in the scaffold's interior. CONCLUSIONS: The harvested chondrocytes from human trachea cartilage expand well in vitro and possess the ability to form new cartilage-like tissue when seeded onto DegraPol matrix. However, improved culture conditions are needed to permit cellular growth throughout cell-polymer constructs.     

Repair of cartilage defects with periosteal grafts.

Alternative sources for repair of cartilage defects are limited and donor sites are associated with morbidity. It is known that cartilage development from periosteal grafts is possible. Various factors have been found positively to affect this process in experimental settings. However, all of these studies were limited to joint cartilage. We conducted an experimental study in rabbits for the investigation of the elastic cartilage regeneration from perichondrial and periosteal grafts together with the effects of hyaluronan on this process. 1 x 1 cm(2) cartilage defects were created on the elastic ear cartilage of rabbits. Four experimental groups with 18 ears in each group were created: Group 1 (repair with perichondrium graft), group 2 (repair with periosteum graft), group 3 (repair with periosteum graft+hyaluronan), group 4 (defect-only control group). Macroscopic and microscopic evaluations were done on the 4th, 8th and 12th weeks. Cellular morphology of the regenerated cartilage and its integration and similarity with adjacent cartilage were evaluated. Cartilage regeneration groups 1, 2 and 3 were found to be statistically different from the control group. There was not a significant difference between groups 1 and 2 or 2 and 3. There is no significant difference between perichondrial and periosteal grafts in cartilage regeneration, and hyaluronan has no beneficial effect on this process.     

Growth of two types of cartilage after implantation of free autogeneic perichondrial grafts

The perichondrium of adult rats was dissected from the posterior side of the ear where a plane of separation can be easily found between the superficial chondrocytes and the rest of the cartilage. When pulled off, the perichondrium brings with it a cartilaginous strip adhered to its inner layer, with the detachment surface showing projections of broken capsular matrix (PBCM). The perichondrium and subperichondrial cartilage were then transferred as autogeneic grafts to preformed muscle pockets of the abdominal wall and to everted vein chambers placed free in the iliac blood flow. During a period of one to 12 days, chondrogenesis was studied in the grafts and in the graft bed areas next to subperichondrial cartilage. When the perichondrium was placed into a muscular pouch, wherein perichondrocytes survived and a prominent vascular ingrowth in the graft bed was observed, the presence of two types of newly formed cartilage was demonstrated (Types I and II). These types showed differences in their location, time of appearance, and microscopic characteristics. Type I neocartilage appeared in the inner layer of the perichondrium on the third or fourth day after grafting; at this time the cells, surrounded by a well-defined capsular matrix, were large, darkly stained, and highly electron dense. Type II neocartilage, separated from Type I by the PBCM, appeared in the graft bed area located within perichondrial folds on the sixth or seventh day after implantation. Their cells showed a poorly defined capsular matrix and were smaller, lighter stained, and less electron dense than those of Type I. When the perichondrium was transplanted to everted vein chambers placed in the iliac blood flow, wherein perichondrocytes survived and vascular ingrowth from the graft bed was not present, Type I neocartilage was formed but Type II was not. The morphologic and histoautoradiographic findings in these studies suggest that Type I cells come from perichondrocytes of the inner perichondrial layer, whereas Type II cells originate from the undifferentiated perivascular mesenchymal cells of the graft bed.     

The culture of articular chondrocytes in hydrogel constructs within a bioreactor enhances cell proliferation and matrix synthesis

Bovine and human articular chondrocytes were seeded in 2% alginate constructs and cultured for up to 19 days in a rotating-wall-vessel (RWV) and under static conditions. Culture within the RWV enhanced DNA levels for bovine chondrocyte-seeded constructs when compared with static conditions but did not produce enhancement for human cells. There was a significant enhancement of glycosaminoglycans and hydroxyproline synthesis for both bovine and human chondrocytes. In all cases, histological analysis revealed enhanced Safranin-O staining in the peripheral regions of the constructs compared with the central region. There was an overall increase in staining intensity after culture within the RWV compared with static conditions. Type-II collagen was produced by both bovine and human chondrocytes in the peripheral and central regions of the constructs and the staining intensity was enhanced by culture within the RWV. A capsule of flattened cells containing type-I collagen developed around the constructs maintained under static conditions when seeded with either bovine or human chondrocytes, but not when cultured within the RWV bioreactor.     

Stabilized autologous fibrin-chondrocyte constructs for cartilage repair in vivo

Stabilization of fibrin-chondrocyte constructs with fibrinolytical inhibitors has been shown to be a feasible method for the reconstruction of cartilage in vitro. In this study, the method was tested in vivo. Autologous cultures were used to form stabilized fibrin-chondrocyte constructs that were injected into auricular cartilage defects of rabbits. Stabilization was achieved by high doses of fibrinolytic inhibitors. Samples were prepared for magnetic resonance imaging, histology, and immunohistochemistry after 1, 2, 4, and 6 months. Defects of the contralateral ear, which were treated with stabilized fibrin without cells, were used for controlled comparisons. In all cell-fibrin samples, cartilage-like tissue was present. Immunohistochemistry revealed the presence of collagen II. This finding was similar for all observations. In the control samples, only minor new cartilage could be detected at the cut edges. The reconstruction of cartilage in vivo by injecting fibrin-chondrocyte constructs, stabilized with inhibitors of fibrinolysis, is thus possible.     

Inhibitory effect of mature cartilage on perichondrial neochondrogenesis

The perichondrium adhering to mature cartilage is not active, but that separated from cartilage is highly chondrogenic. Cartilage formation from isolated perichondrium does not last forever, and perichondrium soon becomes inactive. What activates or inactivates the perichondrium. The authors investigated the effect of mature cartilage on the cartilage formation from perichondrial graft material. The results showed that mature cartilage attached to the perichondrium inhibited neochondrogenesis. The phenomenon that cartilage--a product of chondrogenesis--inhibits neochondrogenesis of perichondrium can be called negative feedback.     

Induction of cartilage growth in a rabbit ear model: a pilot study

Identifying the control of cartilage regeneration is important both clinically and in tissue engineering research. A rabbit ear model was used to simulate surgery and trauma to explore the effect of perichondrial stripping on underlying cartilage in vivo. Ten rabbits (20 ears) formed four groups: two controls and two experimental groups. Group 1 served as the unoperated control group and underwent no treatment. Group 2 served as the operated control group and underwent elevation of auricular skin flaps without stripping the perichondrium. Groups 3 and 4 underwent increasing degrees of surgical insult. Group 3 underwent elevation of a skin flap with stripping of the perichondrium on both sides of the cartilage. Group 4 underwent both perichondrial stripping and the insertion of a thin silicone sheet as a barrier between the denuded cartilage and the skin flaps. At 3 months, punch biopsies of the cartilage were performed in each zone of insult, creating multiple thin sections. The results were analyzed using a computerized morphometry system. Histopathological examination of the groups revealed a regenerative layer of neocartilage which showed distinct hypercellular features of regeneration. The thickness of the new layer was proportional to the degree of the insult (p<0.01). A controlled insult to the perichondrium created a regenerative layer of cartilage; it seems that this layer of neocartilage is proportional to the insult. Further studies are in progress to clarify these findings.     

The role of perichondrium in modifying curved cartilage: an experimental study

A study of 11 strips of curved cartilage harvested from 11 pigs' ears was undertaken to compare quantitatively the forces required to bend the strips before and after modification. The results show no statistical difference between the intact curvature and curvature produced when skin and perichondrium were resutured over 4 full-thickness, cartilaginous crosscuts. These in vitro findings suggest that, when one attempts to modify the shape of curved cartilage, lasting results may be obtained by shortening the convex surface length using a wedge excision method. The perichondrium is incised on the concave surface or the surface that will become longer with cartilage scoring, such as in otoplasty. If the cartilage surface will require elongation for ultimate straightening, the perichondrium as well as the cartilage must be incised.     

Minced cartilage without cell culture serves as an effective intraoperative cell source for cartilage repair.

Traumatic articular cartilage injuries heal poorly and may predispose patients to the early onset of osteoarthritis. One current treatment relies on surgical delivery of autologous chondrocytes that are prepared, prior to implantation, through ex vivo cell expansion of cartilage biopsy cells. The requirement for cell expansion, however, is both complex and expensive and has proven to be a major hurdle in achieving a widespread adoption of the treatment. This study presents evidence that autologous chondrocyte implantation can be delivered without requiring ex vivo cell expansion. The proposed improvement relies on mechanical fragmentation of cartilage tissue sufficient to mobilize embedded chondrocytes via increased tissue surface area. Our outgrowth study, which was used to demonstrate chondrocyte migration and growth, indicated that fragmented cartilage tissue is a rich source for chondrocyte redistribution. The chondrocytes outgrown into 3-D scaffolds also formed cartilage-like tissue when implanted in SCID mice. Direct treatment of full-thickness chondral defects in goats using cartilage fragments on a resorbable scaffold produced hyaline-like repair tissue at 6 months. Thus, delivery of chondrocytes in the form of cartilage tissue fragments in conjunction with appropriate polymeric scaffolds provides a novel intraoperative approach for cell-based cartilage repair.