Efficacy of thermoresponsive,photocrosslinkable hydrogels derived from decellularized tendon and cartilage extracellular matrix for cartilage tissue engineering

Tissue engineering using adult mesenchymal stem cells (MSCs), a promising approach for cartilage repair, is highly dependent on the nature of the matrix scaffold. Thermoresponsive, photocrosslinkable hydrogels were fabricated by functionalizing pepsin‐soluble decellularized tendon and cartilage extracellular matrices (ECM) with methacrylate groups. Methacrylated gelatin hydrogels served as controls. When seeded with human bone marrow MSCs and cultured in chondrogenic medium, methacrylated ECM hydrogels experienced less cell‐mediated contraction, as compared against non‐methacrylated ECM hydrogels. However, methacrylation slowed or diminished chondrogenic differentiation of seeded MSCs, as determined through analyses of gene expression, biochemical composition and histology. In particular, methacrylated cartilage hydrogels supported minimal due to chondrogenesis over 42 weeks, as hydrogel disintegration beginning at day 14 presumably compromised cell–matrix interactions. As compared against methacrylated gelatin hydrogels, MSCs cultured in non‐methacrylated ECM hydrogels exhibited comparable expression of chondrogenic genes (Sox9, Aggrecan and collagen type II) but increased collagen type I expression. Non‐methacrylated cartilage hydrogels did not promote chondrogenesis to a greater extent than either non‐methacrylated or methacrylated tendon hydrogels. Whereas methacrylated gelatin hydrogels supported relatively homogeneous increases in proteoglycan and collagen type II deposition throughout the construct over 42 days, ECM hydrogels possessed greater heterogeneity of staining intensity and construct morphology. These results do not support the utility of pepsin‐solubilized cartilage and tendon hydrogels for cartilage tissue engineering over methacrylated gelatin hydrogels. Methacrylation of tendon and cartilage ECM hydrogels permits thermal‐ and light‐induced polymerization but compromises chondrogenic differentiation of seeded MSCs.     

Co-culture in cartilage tissue engineering

For biotechnological research in vitro in general and tissue engineering specifically, it is essential to mimic the natural conditions of the cellular environment as much as possible. In choosing a model system for in vitro experiments, the investigator always has to balance between being able to observe, measure or manipulate cell behaviour and copying the in situ environment of that cell. Most tissues in the body consist of more than one cell type. The organization of the cells in the tissue is essential for the tissue's normal development, homeostasis and repair reaction. In a co-culture system, two or more cell types brought together in the same culture environment very likely interact and communicate. Co-culture has proved to be a powerful in vitro tool in unravelling the importance of cellular interactions during normal physiology, homeostasis, repair and regeneration. The first co-culture studies focused mainly on the influence of cellular interactions on oocytes maturation to a pre-implantation blastocyst. Therefore, a brief overview of these studies is given here. Later on in the history of co-culture studies, it was applied to study cell-cell communication, after which, almost immediately as the field of tissue engineering was recognized, it was introduced in tissue engineering to study cellular interactions and their influence on tissue formation. This review discusses the introduction and applications of co-culture systems in cell biology research, with the emphasis on tissue engineering and its possible application for studying cartilage regeneration.     

Novel injectable gel (system) as a vehicle for human articular chondrocytes in cartilage tissue regeneration

We developed a novel injectable carrageenan/fibrin/hyaluronic acid‐based hydrogel with in situ gelling properties to be seeded with chondrogenic cells and used for cartilage tissue engineering applications. We first analysed the distribution within the hydrogel construct and the phenotype of human articular chondrocytes (HACs) cultured for 3 weeks in vitro. We observed a statistically significant increase in the cell number during the first 2 weeks and maintenance of cell viability throughout the cell culture, together with the deposition/formation of a cartilage‐specific extracellular matrix (ECM). Taking advantage of a new in vivo model that allows the integration between newly formed and preexisting cartilage in immunodeficient mice to be investigated, we showed that injectable hydrogel seeded with human articular chondrocytes was able to regenerate and repair an experimentally made lesion in bovine articular cartilage, thus demonstrating the potential of this novel cell delivery system for cartilage tissue engineering. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of costal cartilage and its suitability as a cell source for articular cartilage tissue engineering

Costal cartilage is a promising donor source of chondrocytes to alleviate cell scarcity in articular cartilage tissue engineering. Limited knowledge exists, however, on costal cartilage characteristics. This study describes the characterization of costal cartilage and articular cartilage properties and compares neocartilage engineered with costal chondrocytes to native articular cartilage, all within a sheep model. Specifically, we (a) quantitatively characterized the properties of costal cartilage in comparison to patellofemoral articular cartilage, and (b) evaluated the quality of neocartilage derived from costal chondrocytes for potential use in articular cartilage regeneration. Ovine costal and articular cartilages from various topographical locations were characterized mechanically, biochemically, and histologically. Costal cartilage was stiffer in compression but softer and weaker in tension than articular cartilage. These differences were attributed to high amounts of glycosaminoglycans and mineralization and a low amount of collagen in costal cartilage. Compared to articular cartilage, costal cartilage was more densely populated with chondrocytes, rendering it an excellent chondrocyte source. In terms of tissue engineering, using the self‐assembling process, costal chondrocytes formed articular cartilage‐like neocartilage. Quantitatively compared via a functionality index, neocartilage achieved 55% of the medial condyle cartilage mechanical and biochemical properties. This characterization study highlighted the differences between costal and articular cartilages in native forms and demonstrated that costal cartilage is a valuable source of chondrocytes suitable for articular cartilage regeneration strategies.     

Decellularized matrices for tissue engineering

Importance of the field: Biomimetic scaffolds and substrates of extracellular matrices (ECMs) play an important role in the regulation of cell function and in the guidance of new tissue regeneration, as an ECM has the intrinsic cues necessary to communicate with and dictate to cells.

Areas covered in this review: This paper reviews the latest developments in ECM scaffolds and substrates obtained from decellularized tissues, organs or cultured cells and their application in tissue engineering. The ECM composition, structure, interaction with surrounding cells, preparation method and usage in the regeneration of various tissues and organs are summarised.

What the reader will gain: The advantages and challenges of decellularized matrices are highlighted.

Take home message: Similarity in the composition, microstructure and biomechanical properties of the decellularized scaffolds and substrates to those of the native tissues and organs maximizes the promotion effect in the regeneration of both structural and functional tissues and organs. Simple tissues as well as complicated organs have been decellularized and decellularization methods have been optimized to completely remove the cellular components while keeping the ECM intact.     

Effect of double growth factor release on cartilage tissue engineering

The effects of double release of insulin‐like growth factor I (IGF‐I) and growth factor β1 (TGF–β1) from nanoparticles on the growth of bone marrow mesenchymal stem cells and their differentiation into cartilage cells were studied on PLGA scaffolds. The release was achieved by using nanoparticles of poly(lactic acid‐co‐glycolic acid) (PLGA) and poly(N‐isopropylacrylamide) (PNIPAM) carrying IGF‐I and TGF–β1, respectively. On tissue culture polystyrene (TCPS), TGF‐β1 released from PNIPAM nanoparticles was found to have a significant effect on proliferation, while IGF‐I encouraged differentiation, as shown by collagen type II deposition. The study was then conducted on macroporous (pore size 200–400 µm) PLGA scaffolds. It was observed that the combination of IGF‐I and TGF‐β1 yielded better results in terms of collagen type II and aggrecan expression than GF‐free and single GF‐containing applications. It thus appears that gradual release of a combination of growth factors from nanoparticles could make a significant contribution to the quality of the engineered cartilage tissue. Copyright © 2011 John Wiley & Sons, Ltd.     

An additive manufacturing‐based PCL–alginate–chondrocyte bioprinted scaffold for cartilage tissue engineering

Regenerative medicine is targeted to improve, restore or replace damaged tissues or organs using a combination of cells, materials and growth factors. Both tissue engineering and developmental biology currently deal with the process of tissue self‐assembly and extracellular matrix (ECM) deposition. In this investigation, additive manufacturing (AM) with a multihead deposition system (MHDS) was used to fabricate three‐dimensional (3D) cell‐printed scaffolds using layer‐by‐layer (LBL) deposition of polycaprolactone (PCL) and chondrocyte cell‐encapsulated alginate hydrogel. Appropriate cell dispensing conditions and optimum alginate concentrations for maintaining cell viability were determined. In vitro cell‐based biochemical assays were performed to determine glycosaminoglycans (GAGs), DNA and total collagen contents from different PCL–alginate gel constructs. PCL–alginate gels containing transforming growth factor‐β (TGFβ) showed higher ECM formation. The 3D cell‐printed scaffolds of PCL–alginate gel were implanted in the dorsal subcutaneous spaces of female nude mice. Histochemical [Alcian blue and haematoxylin and eosin (H&E) staining] and immunohistochemical (type II collagen) analyses of the retrieved implants after 4 weeks revealed enhanced cartilage tissue and type II collagen fibril formation in the PCL–alginate gel (+TGFβ) hybrid scaffold. In conclusion, we present an innovative cell‐printed scaffold for cartilage regeneration fabricated by an advanced bioprinting technology. Copyright © 2013 John Wiley & Sons, Ltd.     

Effect of visco‐elastic silk–chitosan microcomposite scaffolds on matrix deposition and biomechanical functionality for cartilage tissue engineering

Commonly used polymer‐based scaffolds often lack visco‐elastic properties to serve as a replacement for cartilage tissue. This study explores the effect of reinforcement of silk matrix with chitosan microparticles to create a visco‐elastic matrix that could support the redifferentiation of expanded chondrocytes. Goat chondrocytes produced collagen type II and glycosaminoglycan (GAG)‐enriched matrix on all the scaffolds (silk:chitosan 1:1, 1:2 and 2:1). The control group of silk‐only constructs suffered from leaching out of GAG molecules into the medium. Chitosan‐reinforced scaffolds retained a statistically significant (p < 0.02) higher amount of GAG, which in turn significantly increased (p < 0.005) the aggregate modulus (as compared to silk‐only controls) of the construct akin to that of native tissue. Furthermore, the microcomposite constructs demonstrated highly pronounced hysteresis at 4% strain up to 400 cycles, mimicking the visco‐elastic properties of native cartilage tissue. These results demonstrated a step towards optimizing the design of biomaterial scaffolds used for cartilage tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.     

Comprehensive characterization of chondrocyte cultures in plasma and whole blood biomatrices for cartilage tissue engineering

Many synthetic polymers and biomaterials have been used as matrices for 3D chondrocyte seeding and transplantation in the field of cartilage tissue engineering. To develop a fully autologous carrier for chondrocyte cultivation, we examined the feasibility of allogeneic plasma and whole blood‐based matrices and compared them to agarose constructs. Primary articular chondrocytes isolated from 12‐month‐old pigs were embedded into agarose, plasma and whole blood matrices and cultivated under static‐free swelling conditions for up to four weeks. To evaluate the quality of the synthesized extracellular matrix (ECM), constructs were subjected to weekly examinations using histological staining, spectrophotometry, immunohistochemistry and biochemical analysis. In addition, gene expression of cartilage‐specific markers such as aggrecan, Sox9 and collagen types I, II and X was determined by RT‐PCR. Chondrocyte morphology was assessed via scanning electron microscopy and viability staining, including proliferation and apoptosis assays. Finally, ¹³ C NMR spectroscopy provided further evidence of synthesis of ECM components. It was shown that chondrocyte cultivation in allogeneic plasma and whole‐blood matrices promoted sufficient chondrocyte viability and differentiation behaviour, resulting in neo‐formation of a hyaline‐like cartilage matrix. Copyright © 2012 John Wiley & Sons, Ltd.     

The application of POSS nanostructures in cartilage tissue engineering: the chondrocyte response to nanoscale geometry

Despite extensive research into cartilage tissue engineering (CTE), there is still no scaffold ideal for clinical applications. Various synthetic and natural polymers have been investigated in vitro and in vivo, but none have reached widespread clinical use. The authors investigate the potential of POSS–PCU, a synthetic nanocomposite polymer, for use in CTE. POSS–PCU is modified with silsesquioxane nanostructures that improve its biological and physical properties. The ability of POSS–PCU to support the growth of ovine nasoseptal chondrocytes was evaluated against a polymer widely used in CTE, polycaprolactone (PCL). Scaffolds with varied concentrations of the POSS molecule were also synthesized to investigate their effect on chondrocyte growth. Chondrocytes were seeded onto scaffold disks (PCU negative control; POSS–PCU 2%, 4%, 6%, 8%; PCL). Cytocompatibilty was evaluated using cell viability, total DNA, collagen and GAG assays. Chondrocytes cultured on POSS–PCU (2% POSS) scaffolds had significantly higher viability than PCL scaffolds (p < 0.001). Total DNA, collagen and sGAG protein were also greater on POSS–PCU scaffolds compared with PCL (p > 0.05). POSS–PCU (6% and 8% POSS) had improved viability and proliferation over an 18 day culture period compared with 2% and 4% POSS–PCU (p < 0.0001). Increasing the percentage of POSS in the scaffolds increased the size of the pores found in the scaffolds (p < 0.05). POSS–PCU has excellent potential for use in CTE. It supports the growth of chondrocytes in vitro and the POSS modification significantly enhances the growth and proliferation of nasoseptal chondrocytes compared with traditional scaffolds such as PCL. Copyright © 2013 John Wiley & Sons, Ltd.     

RHEB: a potential regulator of chondrocyte phenotype for cartilage tissue regeneration

As articular cartilage has a limited ability to self‐repair, successful cartilage regeneration requires clinical‐grade chondrocytes with innate characteristics. However, cartilage regeneration via chondrocyte transplantation is challenging, because chondrocytes lose their innate characteristics during in vitro expansion. Here, we investigated the mechanistic underpinning of the gene Ras homologue enriched in brain (RHEB) in the control of senescence and dedifferentiation through the modulation of oxidative stress in chondrocytes, a hallmark of osteoarthritis. Serial expansion of human chondrocytes led to senescence, dedifferentiation and oxidative stress. RHEB maintained the innate characteristics of chondrocytes by regulating senescence, dedifferentiation and oxidative stress, leading to the upregulation of COL2 expression via SOX9 and the downregulation of p27 expression via MCL1. RHEB also decreased the expression of COL10. RHEB knockdown mimics decreased the expression of SOX9, COL2 and MCL1, while abrogating the suppressive function of RHEB on p27 and COL10 in chondrocytes. RHEB‐overexpressing chondrocytes successfully formed cartilage tissue in vitro as well as in vivo, with increased expression of GAG matrix and chondrogenic markers. RHEB induces a distinct gene expression signature that maintained the innate chondrogenic properties over a long period. Therefore, RHEB expression represents a potentially useful mechanism in terms of cartilage tissue regeneration from chondrocytes, by which chondrocyte phenotypic and molecular characteristics can be retained through the modulation of senescence, dedifferentiation and oxidative stress. Copyright © 2016 John Wiley & Sons, Ltd.     

Mechanical compression of articular cartilage induces chondrocyte proliferation and inhibits proteoglycan synthesis by activation of the ERK pathway: implications for tissue engineering and regenerative medicine

Articular cartilage is recalcitrant to endogenous repair and regeneration and is thus a focus of tissue engineering and regenerative medicine strategies. A prerequisite for articular cartilage tissue engineering is an understanding of the signal transduction pathways involved in mechanical compression during trauma or disease. We sought to explore the role of the extracellular signal‐regulated kinase 1/2 (ERK 1/2) pathway in chondrocyte proliferation and proteoglycan synthesis following acute mechanical compression. Bovine articular cartilage explants were cultured with and without the ERK 1/2 pathway inhibitor PD98059. Cartilage explants were statically loaded to 40% strain at a strain rate of 1/s for 5 s. Control explants were cultured under similar conditions but were not loaded. There were four experimental groups: (a) no load, without inhibitor; (b) no load, with the inhibitor PD98059; (c) loaded, without the inhibitor; and (d) loaded, with the inhibitor PD98059. The explants were cultured for varying durations from 5 min to 5 days and were then analysed by biochemical and immunohistochemical methods. Mechanical compression induced phosphorylation of ERK 1/2, and this was attenuated with the ERK 1/2 pathway inhibitor PD98059 in a dose‐dependent manner. Chondrocyte proliferation was increased by mechanical compression. This effect was blocked by the inhibitor of the ERK 1/2 pathway. Mechanical compression also led to a decrease in proteoglycan synthesis that was reversed with inhibitor PD98059. In conclusion, the ERK 1/2 pathway is involved in the proliferative and biosynthetic response of chondrocytes following acute static mechanical compression. Copyright © 2009 John Wiley & Sons, Ltd.     

Evaluation of the potential of novel PCL-PPDX biodegradable scaffolds as support materials for cartilage tissue engineering

Cartilage is a specialized tissue represented by a group of particular cells (the chondrocytes) and an abundant extracellular matrix. Because of the reduced regenerative capacity of this tissue, cartilage injuries are often difficult to handle. Nowadays tissue engineering has emerged as a very promising discipline, and biodegradable polymeric scaffolds are widely used as tissue supports. In cartilage injuries, the use of autologous chondrocyte implantation from non-affected cartilage zones has emerged as a very interesting technique, where chondrocytes are expanded in order to obtain a greater number of cells. Nevertheless, it has been reported that chondrocytes in bidimensional cultures suffer a dedifferentiation process. The present study sought, in the first place, to standardize a novel protocol in order to obtain primary cultures of chondrocytes from newborn rabbit hyaline cartilage from the xiphoid process. Second, the potential of porous three-dimensional (3D) biodegradable polymeric matrices as support materials for chondrocytes was evaluated: a novel poly(ε-caprolactone)-poly(p-dioxanone) (PCL-PPDX) blend in a 90:10 w:w ratio and poly(ε-caprolactone) (PCL). After achieving the standardization, a typical round-shaped chondrocyte morphology and the expression of collagen type II and aggrecan, evaluated by RT-PCR, were observed. Second-passage chondrocytes adhered effectively to these scaffolds, although cell growth at 7 days in culture was significantly less in the PCL-PPDX blend. After 3 weeks of culture on PCL-PPDX or PCL, the cells expressed collagen type II. The present study demonstrates the potential, unknown until now, of PCL-PPDX blend scaffolds in the field of cartilage tissue engineering.     

组织工程软骨与柚皮苷联合修复兔膝关节软骨缺损的组织学证实

背景：目前临床上虽有多种方法用于治疗软骨缺损,但没有从根本上解决关节软骨缺损修复问题。目的：通过组织学研究进一步评价柚皮苷结合组织工程软骨修复兔关节软骨缺损的效果。方法：取兔骨髓间充质干细胞体外增殖后,复合于改建后的脱细胞真皮基质载体上,制成组织工程软骨,植入到兔膝关节软骨缺损,并以柚皮苷汤灌胃,于4,8周后分别对修复组织进行苏木精-伊红、Masson三色染色、甲苯胺蓝染色、Ⅱ型胶原染色、Ⅹ型胶原染色等组织学检查。结果与结论：术后8周,柚皮苷结合干细胞复合体组缺损处修复组织变成乳白色,半透明光滑组织,缺损修复组织与周围正常软骨已基本难区分,表面光滑。组织学检查发现修复缺损处基本为新生软骨填充。结果证实,柚皮苷结合组织工程软骨能提高家兔膝关节软骨缺损的修复质量。     

The use of a novel bone allograft wash process to generate a biocompatible,mechanically stable and osteoinductive biological scaffold for use in bone tissue engineering

Fresh‐frozen biological allograft remains the most effective substitute for the ‘gold standard’ autograft, sharing many of its osteogenic properties but, conversely, lacking viable osteogenic cells. Tissue engineering offers the opportunity to improve the osseointegration of this material through the addition of mesenchymal stem cells (MSCs). However, the presence of dead, immunogenic and potentially harmful bone marrow could hinder cell adhesion and differentiation, graft augmentation and incorporation, and wash procedures are therefore being utilized to remove the marrow, thereby improving the material's safety. To this end, we assessed the efficiency of a novel wash technique to produce a biocompatible, biological scaffold void of cellular material that was mechanically stable and had osteoinductive potential. The outcomes of our investigations demonstrated the efficient removal of marrow components (~99.6%), resulting in a biocompatible material with conserved biomechanical stability. Additionally, the scaffold was able to induce osteogenic differentiation of MSCs, with increases in osteogenic gene expression observed following extended culture. This study demonstrates the efficiency of the novel wash process and the potential of the resultant biological material to serve as a scaffold in bone allograft tissue engineering. © 2014 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons Ltd.     

Decellularized caprine liver extracellular matrix as a 2D substrate coating and 3D hydrogel platform for vascularized liver tissue engineering

Development of a vascularized liver tissue construct is a need of an hour to circumvent the current demand of liver transplantation in health care sector. An appropriate matrix must support liver cell viability, functionality, and development of microvasculature. With this perspective, here, we report the use of decellularized caprine liver extracellular matrix (CLECM) derived hydrogel for tissue engineering applications. First, CLECM was used as a substrate coating material for 2D hepatocyte culture. HepG2 cells cultured on CLECM‐coated surface showed higher albumin, urea, glycogen, and GAGs synthesis in comparison with collagen‐coated surface (taken as control for the study). Thereafter, the cells were encapsulated in CLECM hydrogels for 3D culture. In CLECM hydrogels, HepG2 cells showed highly differentiated and polarized phenotype with the appearance of bile canaliculi‐like structures and enhanced expression of mature hepatocyte markers. We further showed that CLECM hydrogels also supported the development of microvasculature in vitro, thus making it a suitable candidate for development of a prevascularized liver tissue construct. In conclusion, we proved the superiority of CLECM over collagen for 2D/3D human hepatocyte and endothelial cell culture. CLECM could serve as an efficient biomaterial platform in the development of a liver tissue construct for application in tissue engineering.     

Tissue engineering: chondrocyte culture on type 1 collagen support. Cytohistological and immunohistochemical study

The scope of our study is to evaluate the possibility of cultivating and expanding human chondrocytes and seeding them on pure equine type I collagen support. Our results show that human articular cartilaginous cells can multiply and grow on type I collagen substrate with production of extracellular matrix. This type of chondrocyte culture on a support can be used for repairing cartilaginous lesions since they show a correct morphology (evaluated by cytological and histological methods) and a suitable differentiation and phenotype as shown by Alcian PAS staining to indicate the presence of mucopolysaccharides, and immunohistochemical methods to identify collagen II. We believe that these chondrocyte cultures on this biomaterial can be used for repairing cartilaginous lesions with improvement of surgical technique; the support allows adhesion of the chondrocytes to the cartilaginous lesion and a mallebility that favours optimum spatial adaptation.     

Influence of mesenchymal stem cells on stomach tissue engineering using small intestinal submucosa

Small intestinal submucosa (SIS) is a biodegradable collagen‐rich matrix containing functional growth factors. We have previously reported encouraging outcomes for regeneration of an artificial defect in the rodent stomach using SIS grafts, although the muscular layer was diminutive. In this study, we investigated the feasibility of SIS in conjunction with mesenchymal stem cells (MSCs) for regeneration of the gastrointestinal tract. MSCs from the bone marrow of green fluorescence protein (GFP)‐transgenic Sprague–Dawley (SD) rats were isolated and expanded ex vivo. A 1 cm whole‐layer stomach defect in SD rats was repaired using: a plain SIS graft without MSCs (group 1, control); a plain SIS graft followed by intravenous injection of MSCs (group 2); a SIS graft co‐cultured with MSCs (group 3); or a SIS sandwich containing an MSC sheet (group 4). Pharmacological, electrophysiological and immunohistochemical examination was performed to evaluate the regenerated stomach tissue. Contractility in response to a muscarinic receptor agonist, a nitric oxide precursor or electrical field stimulation was observed in all groups. SIS grafts seeded with MSCs (groups 3 and 4) appeared to support improved regeneration compared with SIS grafts not seeded with MSCs (groups 1 and 2), by enabling the development of well‐structured smooth muscle layers of significantly increased length. GFP expression was detected in the regenerated interstitial tissue, with fibroblast‐like cells in the seeded‐SIS groups. SIS potently induced pharmacological and electrophysiological regeneration of the digestive tract, and seeded MSCs provided an enriched environment that supported tissue regeneration by the SIS graft in the engineered stomach. © 2013 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.     

耻骨联合可作为组织工程种子细胞的供区？

背景：耻骨联合是与关节盘、半月板或椎间盘具有相同性质的组织，但是将其作为组织工程种子细胞供区的研究，至今鲜有报道。目的：观察耻骨联合作为组织工程种子细胞供区的可行性。方法：取大鼠的耻骨联合组织进行苏木精-伊红、阿尔新蓝及Ⅱ型胶原免疫组织化学染色，观察细胞外基质的组织学特征。再将分离自大鼠耻骨联合组织的细胞，在体外培养扩增后，与藻酸盐凝胶支架复合立体培养，通过活细胞荧光标记方法，观察细胞存活率，通过21d长期培养观察细胞增殖情况。测量8具成年男性骨盆标本的耻骨联合软骨区的体积，判断其是否有利于分离足够数量的种子细胞。结果与结论：苏木精-伊红染色显示，大鼠耻骨联合组织有大量平行或交叉排列的纤维束，细胞单独散在、成对或成行排列分布于纤维束之间的陷窝内。阿尔新蓝染色和Ⅱ型胶原免疫组织化学染色发现，大鼠耻骨联合组织广泛着色，细胞及陷窝附近着色较深。藻酸盐凝胶支架为多孔结构，孔隙间交互通连，孔隙直径为（27．0±16．7）pm，孔隙壁较光滑。在支架上细胞存活率为（72．4±4．5）％。在体外长期培养过程中，支架上的细胞呈圆球形，逐渐形成“同源细胞群”，培养13d时观察到的细胞团，在第21天时明显增大，所含细胞数明显增多。成年男性耻骨联合组织的体积为（1．13±0．21）cm3。结果证实，大鼠耻骨联合组织细胞具有软骨细胞的排列特征，细胞外间质有软骨组织特异性基质成分。取自大鼠耻骨联合的细胞，在体外立体培养具有持续的增殖能力，耻骨联合组织可作为软骨组织工程种子细胞供区。成年男性耻骨联合组织量较大，有利于分离获取较多的原代细胞。     

Cartilage tissue engineering on macroporous scaffolds using human tooth germ stem cells

The main objective was to study cartilage regeneration through differentiation of human tooth germ stem cells (HTGSCs) into chondrocytes on different three‐dimensional (3D) scaffolds (PCL, PLLA and PCL–PLLA). Scaffold topographies were studied by scanning electron microscopy and it was found that the scaffolds had interconnected macroporous structures. HTGSCs were isolated from impacted third molar tooth germs of young adult patients and grown for 3 weeks on the scaffolds in chondrogenic differentiation medium. Cell proliferation on the scaffolds was determined by MTS assay and it was observed that all scaffolds supported cell proliferation. Immunostaining was carried out for morphological and differentiation analyses. Immunohistochemical analyses revealed that the cells attached onto the scaffolds and deposited cartilage‐specific extracellular matrix (ECM). Real‐time PCR was performed to determine the expression levels of cartilage‐specific genes. After 21 days of incubation in cartilage differentiation medium, expression of collagen type II increased only in the cells seeded onto PCL–PLLA blend scaffolds. Similarly, aggrecan expression was the highest on PCL–PLLA scaffolds after 3 weeks. These results suggest that all the scaffolds, and especially PCL–PLLA, were suitable for chondrogenic differentiation of HTGSCs. Copyright © 2015 John Wiley & Sons, Ltd.