Collagen Scaffolds in Cartilage Tissue Engineering and Relevant Approaches for Future Development

BACKGROUND: Cartilage tissue engineering (CTE) aims to obtain a structure mimicking native cartilage tissue through the combination of relevant cells, three-dimensional scaffolds, and extraneous signals. Implantation of ‘matured’ constructs is thus expected to provide solution for treating large injury of articular cartilage. Type I collagen is widely used as scaffolds for CTE products undergoing clinical trial, owing to its ubiquitous biocompatibility and vast clinical approval. However, the long-term performance of pure type I collagen scaffolds would suffer from its limited chondrogenic capacity and inferior mechanical properties. This paper aims to provide insights necessary for advancing type I collagen scaffolds in the CTE applications. METHODS: Initially, the interactions of type I/II collagen with CTE-relevant cells [i.e., articular chondrocytes (ACs) and mesenchymal stem cells (MSCs)] are discussed. Next, the physical features and chemical composition of the scaffolds crucial to support chondrogenic activities of AC and MSC are highlighted. Attempts to optimize the collagen scaffolds by blending with natural/synthetic polymers are described. Hybrid strategy in which collagen and structural polymers are combined in non-blending manner is detailed. RESULTS: Type I collagen is sufficient to support cellular activities of ACs and MSCs; however it shows limited chondrogenic performance than type II collagen. Nonetheless, type I collagen is the clinically feasible option since type II collagen shows arthritogenic potency. Physical features of scaffolds such as internal structure, pore size, stiffness, etc. are shown to be crucial in influencing the differentiation fate and secreting extracellular matrixes from ACs and MSCs. Collagen can be blended with native or synthetic polymer to improve the mechanical and bioactivities of final composites. However, the versatility of blending strategy is limited due to denaturation of type I collagen at harsh processing condition. Hybrid strategy is successful in maximizing bioactivity of collagen scaffolds and mechanical robustness of structural polymer. CONCLUSION: Considering the previous improvements of physical and compositional properties of collagen scaffolds and recent manufacturing developments of structural polymer, it is concluded that hybrid strategy is a promising approach to advance further collagen-based scaffolds in CTE.

Effects of extracellular acidosis on pyroptosis of rat articular chondrocytes and its possible mechanisms / 中国药理学通报

Aim To study the effects of extracellular acidosis on articular chondrocytes pyroptosis and its possible mechanisms. Methods Primary articular chondrocytes were incubated in different pH and NAC. The expression of proinflammatory cytokines IL-1β, IL-18, ASC, NLRP3, caspase-1 were detected by Western blot and real-time PCR. The state of pyropto-sis was identified by AO/EB staining and LDH con-tents. The expression of ROS was observed by DCFH-DA, and ELISA was used to detect the IL-1β,IL-18 in cultured supernatants. Results Compared with the normal cell, extracellular acidosis could increase the expression of IL-1β, IL-18, ASC, NLRP3 and caspase-1 , upregulate the fluorescence intensity of in-tercellular ROS, accompanied with the promoted release of LDH. Moreover, it is observed that extra-cellular acidosis could also induce chondrocytes death by AO/EB staining. NAC,the scavenger of ROS could inhibit these effects of extracellular acidosis on chon-drocytes. Conclusion Extracellular acidosis may in-duce chondrocyte pyroptosis via upregulating the intra-cellular ROS content.

Role of acid-sensing ion channel 1 a in matrix turnover and MAPK expression of articular chondrocytes of rats / 中国药理学通报

Aim TostudytheroleofASIC1aonthe matrix turnover and MAPK expression of the rat articu-lar chondrocytes with extracellular acidosis. Methods ArticularchondrocyteswereisolatedfromSprague-Dawley rats, and their phenotype was determined by toluidine blue and immunocytochemical staining. The GAG content of cell culture supernatant was deter-mined by dimethyl-methylene blue spectrophotometric assay, while Hyp content by chloramine T assay. ELISA assay was used to measure MMP-2 , TIMP-2 content. Furthermore, the ERK1/2, p38 MAPK phos-phorylation protein expression levels were tested by Westernblotassay.Results ASIC1acontributedto the effect of GAG, Hyp and TIMP-2 levels reduction induced by extracellular acidification, while the effect of MMP-2 was weaker. Moreover, ASIC1a could in-crease the ERK1/2 , p38 MAPK phosphorylation pro-teinexpressionlevels.Conclusion ASIC1acould regulate rat articular chondrocytes matrix turnover via ERK1/2 and p38 MAPK signaling pathway, and there-by inhibit the rat articular cartilage damage induced by acidosis.

Research of co-culture of adipose derived stem cells and chondrocytes in different culture conditions / 中国矫形外科杂志

[Objective]To investigate the feasibility and optimal condition of in vitro chondrogenesis by co-culture of adipose derived stem cell(ADSC)and chondrocytes in pathologic state so as to provide a new clinical approach for repairing damaged articular cartilage.[Method]ADSC and aricular chondrocytes from osteoarthritis model in adult New Zealand white rabbits were in vitro expanded and then seeded on plate and plug-in type millicell dish respectively.The co-culture cells were cultured in different conditions including different serum concentration(10% FBS and 2% FBS)and dimensions(fibrin scaffold and monolayer).The culture medium was changed every 3 days.The shape of ADSC before and after co-culture was observed by inverted microscope and transmission electron microscope.The expression of aggrecan and type Ⅱ collagen genes in ADSC were studied by toluidine blue staining and immunohistochemistry after in vitro co-culture for 14 days.And the transcription of aggrecan and type Ⅱ collagen genes in ADSC were studied by RT-PCR.[Result]The shape of ADSC was fibroblast-like cells morphologically.And they became round at 7 days after in vitro co-culture.At 14 days after co-culture with chondrocytes,ADSC was changed to chondrocyte-like cells morphologically and increased immunostaining particles of type Ⅱ collagen and enhanced toluidine blue staining.And the transcription of type Ⅱ collagen and aggrecan genes was also increased,especially in the ADSC cultured in fibrin scaffold with 10% FBS.[Conclusion]Chondrocytes in pathologic state may provide chondrogenic microenvironment to induce chondrogenic differentiation of ADSC in vitro,which will be enhanced by high serum concentration and three-dimensional culture.

Studies on repair of rabbit articular cartilage defect by chondrocytes infected by adenovirus vector mediated TGF-?_1 Genes / 中国矫形外科杂志

[Objective]To investigate the effect of transforming growth factor ?1(TGF-?1) genes transfection on the proliferation and differentiation of chondrocyte by adenovirus vector,and to observe the quality in repair cartilage defect through constructing tissue-engineered cartilage.[Method]Using replicat ion defective adenovirus Adeno-X~(TM) as a carrier,Articular chondrocytes were transferred by high titers level recombinant adenovirus taking TGF-?1 genes.Ultromicrostructure,prolife ration anddifferentiation were observed by light and electron microscope,flow cytometry and so on.Induce expression of exogenous genes coding protein and cartilage cell phenotype were detected by Immunocytochemistry and Northern blot.Gross observation,histology and effect classification were used to evaluate results in repair cartilage defect by combi nation of infected chondrocytes and bone matrix gelatin(BMG).[Result]Immunocytochemistric staining showedthe expression of exogenous gene coding proteins after infection,and procollagen Ⅱ mRNA in cells was detected increasingly by Northern blot.TGF-?1 stimulated proliferation of chondrocytes in primary cultured,and resulted in recruitment of articular chondrocytes into S-G2/M phase.Chondrocytes seeded onto BMG showed high level of proliferation.Transplantation in vivo showed: cartilage defect was repaired satisfactorily by Combination of BMG and infected chondrocytes,and the structure of repair tissue got close to normal articular cartilage.[Conclusion] Articular chondrocytes prolifera tion could be stimulated by adenovirus vector taking TGF-?1 genes,which could be used to constructed tissue-engineered cartilage,and applied in repairing joint cartilage defect.

Effects of ageing and arthritic disease on nitric oxide production by human articular chondrocytes

Nitric oxide (NO) has been considered as an important mediator in inflammatory phases and in loss of cartilage. In inflammatory arthritis, NO levels are correlated with disease activity and articular cartilage is able to produce large amounts of NO with the appropriate inducing factor such as cytokines. The old animals are shown to have a greater sensitivity to NO than young animals. This study evaluated the basal production of NO in normal and OA-affected chondroyctes from young and old patients and compared the levels of NO formation in response to IL-1beta. The results showed that the basal levels were 7-fold higher in old chondrocytes than those of young cells. However, the IL-1beta induced NO production was seen to decrease with age. Aminoguianidine (AG), a competitive inhibitor of iNOS, inhibited NO formation completely in both chondrocytes from young and old individuals. However, at the same concentration of AG it caused partial inhibition of NO and iNOS formation in chondrocytes from OA-affected individuals. In addition, although the IL-1beta induced NO production was much lesser than that of young chondrocytes, the inhibition of collagen production by IL-1beta was prominent in old chondrocytes and OA-affected chondrocytes. These results suggest that age-related differences in the regulation of NO production and collagen production, which may affect the ageing cells and osteoarthritic changes in some way.

Effects of ageing and arthritic disease on nitric oxide production by human articular chondrocytes

Nitric oxide (NO) has been considered as an important mediator in inflammatory phases and in loss of cartilage. In inflammatory arthritis, NO levels are correlated with disease activity and articular cartilage is able to produce large amounts of NO with the appropriate inducing factor such as cytokines. The old animals are shown to have a greater sensitivity to NO than young animals. This study evaluated the basal production of NO in normal and OA-affected chondroyctes from young and old patients and compared the levels of NO formation in response to IL-1beta. The results showed that the basal levels were 7-fold higher in old chondrocytes than those of young cells. However, the IL-1beta induced NO production was seen to decrease with age. Aminoguianidine (AG), a competitive inhibitor of iNOS, inhibited NO formation completely in both chondrocytes from young and old individuals. However, at the same concentration of AG it caused partial inhibition of NO and iNOS formation in chondrocytes from OA-affected individuals. In addition, although the IL-1beta induced NO production was much lesser than that of young chondrocytes, the inhibition of collagen production by IL-1beta was prominent in old chondrocytes and OA-affected chondrocytes. These results suggest that age-related differences in the regulation of NO production and collagen production, which may affect the ageing cells and osteoarthritic changes in some way.

Immunohistochemical Reaction of Calcitonin-gene-related Peptide and Type II Collagen and Morphological Changes of Cartilage Implants and Cultured Chondrocytes / 대한해부학회지

The purpose of this study was to investigate the immunohistochemical reaction of calcitonin-gene-related peptide (CGRP) and type II collagen and also morphological changes of cartilage implants and cultured chondrocytes isolated from the articular and costal cartilages. The chondrocytes were isolated from the head of the femur and the 11th costal cartilage of the 6 months old rabbits. De novo implants were prepared from the chondrocytes cultured on the perichondrium by culturing isolated articular chondrocytes. Cultured chondrocytes and implants were evaluated by immunohistochemical staining of CGRP and type II collagen and electron microscopy. Articular chondrocytes maintained the typical phenotype in the 1st and 2nd subcultures, but the costal chondrocytes were transformed into fibroblast-like cells. The articular chondrocytes cultured on the perichondrium were more flattened and formed the cartilage. Most chondrocytes were no loss of type II collagen immunostaining by culturing. Implants replaced by the cultured articular chondrocytes were generally increased CGRP and decreased type II collagen immunoreaction. Electron microscopically the cultured articular chondrocytes had a large euchromatic nucleus, a few granules, and abundant vesicles. During culture, the nucleus became atropy and the cytoplasm contained many large vacuoles. The chondrocytes cultured on the perichondrium showed a lot of segmented rough endoplasmic reticulum and fine short microvilli. During culture, articular chondrocytes maintained typical phenotype and type II collagen reaction. The cultured articular chondrocytes had some organelles and euchromatic nucleus with prominent nucleolus. The chondrocytes cultured on the perichondrium showed active secretion of the matrix with small vesicles and well developed endoplasmic reticulum. The implanted articular chondrocytes showed the decrease of their organelles after secretion of the marix and became increased CGRP and decreased type II collagen immunoreaction.