BMP2 initiates chondrogenic lineage development of adult human mesenchymal stem cells in high-density culture

Human bone marrow-derived mesenchymal stem cells (MSCs) have been shown to differentiate into distinct mesenchymal tissues including bone and cartilage. The capacity of MSCs to replicate undifferentiated and to mature into cartilaginous tissues suggests these cells as an attractive cell source for cartilage tissue engineering. Here we show that the stimulation of human bone marrow-derived MSCs with recombinant bone morphogenetic protein-2 (BMP2) results in chondrogenic lineage development under serum-free conditions. Histological staining of proteoglycan with Alcian blue and immunohistochemical staining of cartilage-specific type II collagen revealed the deposition of typical cartilage extracellular matrix components. Semi-quantitative real-time gene expression analysis of characteristic chondrocytic matrix genes, such as cartilage link protein, cartilage oligomeric matrix protein, aggrecan, and types I, II, and IX collagen, confirmed the induction of the chondrocytic phenotype in high-density culture upon stimulation with BMP2 and transforming growth factor-beta3 (TGFbeta3). Histologic staining of mineralized extracellular matrix with von Kossa, immunostaining of type X collagen (typical for hypertrophic chondrocytes), and gene expression analysis of osteocalcin and adipocyte-specific fatty acid binding protein (aP2) further documented that BMP2 induced chondrogenic lineage development and not osteogenesis and/or adipogenesis in human MSCs. These results suggest BMP2 as a promising candidate for tissue engineering approaches regenerating articular cartilage on the basis of mesenchymal progenitors from bone marrow.     

软骨寡聚基质蛋白过表达对BMP-2诱导骨髓间充质干细胞分化的影响

目的：研究软骨寡聚基质蛋白（cartilage oligomeric matrix protein,COMP）过表达对BMP-2诱导骨髓间充质干细胞成骨及成软骨分化的影响。方法：BMP-2诱导骨髓间充质干细胞分化,通过脂质体转染含人COMP基因的质粒使骨髓间充质干细胞过表达COMP,采用实时定量PCR和Western blotting分析COMP基因过表达、成骨相关基因Ⅰ型胶原、RUNX2、骨钙蛋白以及成软骨相关基因Ⅱ型胶原、SOX9、蛋白聚糖、X型胶原的表达变化;通过茜素红染色观察成骨终末阶段矿化结节的生成情况,阿利新蓝染色观察细胞基质蛋白多糖的合成情况。结果：质粒转染后骨髓间充质干细胞COMP基因蛋白和mRNA表达水平显著提高（P<0.05）。COMP基因过表达后,成骨标记基因RUNX2、Ⅰ型胶原（Col1a1）mRNA水平均显著低于对照组（P<0.05）,RUNX2、骨钙蛋白（Osteocalcin）蛋白表达水平明显低于对照组（P<0.05）,而成软骨标记基因SOX9、蛋白聚糖（Aggrecan）mRNA水平均显著高于对照组（P<0.05）,SOX9、Ⅱ型胶原（Col2a1）蛋白表达均明显多于对照组（P<0.05）。细胞成骨茜素红染色弱于对照组,而阿利新蓝染色强于对照组。过表达组细胞X型胶原（Col10a1）基因表达显著低于对照组（P<0.05）,结论：骨髓间充质干细胞COMP基因过表达可抑制BMP-2诱导其成骨分化,促进骨髓间充质干细胞成软骨分化,并抑制软骨细胞的成熟肥大,为软骨组织工程研究提供新的方向。     

甲状旁腺素促进小鼠软骨细胞成软骨和抑制其终末期分化的实验研究

目的探讨甲状旁腺素(PTH)对小鼠软骨细胞成软骨性的促进作用和终末期分化的抑制作用。方法分离和培养新生小鼠胸骨软骨细胞,经PTH处理,倒置显微镜观察细胞形态的变化;Alcian蓝染色和碱性磷酸酶(ALP)染色方法检测软骨细胞蛋白多糖和ALP的分泌;RT-PCT法和Western blot方法检测细胞内成软骨因子和病理性肥大分化因子基因和蛋白的表达。结果新生小鼠胸骨软骨细胞具有自发成熟分化的特征,与对照组相比,经PTH处理的细胞更接近于软骨细胞形态;PTH明显提高软骨细胞Alcian蓝染色的强度,降低ALP染色的强度;PTH显著提高细胞内Sox9和Aggrecan基因和蛋白的表达,明显降低ALP和Runx2基因和蛋白的表达。结论 PTH具有促进小鼠软骨细胞成软骨和抑制其终末期分化的作用。     

富血小板纤维蛋白对兔骨髓间充质干细胞成软骨分化的影响

目的：观察自体富血小板纤维蛋白（platelet-rich fibrin,PRF）对体外培养的兔骨髓间充质干细胞（Bonemarrowmesenchymalstemcells,BMSCs）成软骨分化的影响。方法：兔心脏采血制备PRF,电镜观察其超微结构;分离培养兔BMSCs,取第3代细胞用于实验．分为PIuF组、阳性对照组、空白对照组。诱导培养21d后,对三组细胞分别进行形态学观察,成软骨鉴定染色（甲苯胺蓝、Ⅱ型胶原免疫组化染色）,软骨相关基因表达检测（Ⅱ型胶原、Aggrecan、SOX9）。结果：PRF组和阳性对照组中BMSCs经诱导后,细胞由长梭形变为三角形、多角形、圆形;甲苯胺蓝、Ⅱ型胶原免疫组化染色均为阳性;Ⅱ型胶原、Aggrecan、SOX9基因表达水平均较高,两组比较无统计学差异,空白对照组未见相关分化现象。结论：PRF在体外可促进兔BMSCs成软骨分化,可作为自体生物材料,在构建组织工程软骨中发挥更好的作用。     

Cartilaginous tissue formation from bone marrow cells using rotating wall vessel (RWV) bioreactor

This is the first successful report of the rapid regeneration of three-dimensional large and homogeneous cartilaginous tissue from rabbit bone marrow cells without a scaffold using a rotating wall vessel (RWV) bioreactor, which simulates a microgravity environment for cells. Bone marrow cells cultured for 3 weeks in DMEM were resuspended and cultured for 4 weeks in the chondrogenic medium within the vessel. Large cylindrical cartilaginous tissue with dimensions of (1.25 +/- 0.06) x (0.60 +/- 0.08) cm (height x diameter) formed. Their cartilage marker expression was confirmed by mRNA expressions of aggrecan, collagen type I and II, and glycosaminoglycan (GAG)/DNA ratio. Their cartilaginous properties were demonstrated by toluidine blue, safranin-O staining, and polarization.     

Chondrogenesis enhanced by overexpression of sox9 gene in mouse bone marrow-derived mesenchymal stem cells

We investigated chondrogenesis of cell-mediated sox9 gene therapy as a new treatment regimen for cartilage regeneration. pIRES2-EGFP vector containing a full-length mouse sox9 cDNA was transfected into bone marrow-derived mesenchymal stem cells (MSCs) by lipofection and chondrogenic differentiation of these cells was evaluated. In vitro high density micromass culture of these sox9 transfected MSCs demonstrated that a matrix-rich micromass aggregate with EGFP expressing MSCs was positively stained by Alcian blue and type II collagen. Next, sox9 transfected MSCs were loaded into the diffusion chamber and transplanted into athymic mice to analyze in vivo chondrogenesis. A massive tissue formation in about 2mm diameter was visible in the chamber after 4 weeks transplantation. Histological examinations demonstrated that both Alcian blue and type II collagen were positively stained in the extracellular matrix of the mass while type X collagen was not stained. These results indicated that cell-mediated sox9 gene therapy could be a novel strategy for hyaline cartilage damage.     

Implication of C-type natriuretic peptide-3 signaling in glycosaminoglycan synthesis and chondrocyte hypertrophy during TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells

This study investigated the involvement of CNP-3, chick homologue for human C-type natriuretic peptide (CNP), in TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells (MSCs). Chondrogenic differentiation of MSCs in pellet cultures was induced by TGF-β1. Chondrogenic differentiation and glycosaminoglycan synthesis were analyzed on the basis of basic histology, collagen type II expression, and Alcian blue staining. Antibodies against CNP and NPR-B were used to block their function during these processes. Results revealed that expression of CNP-3 and NPR-B in MSCs were regulated by TGF-β1 in monolayer cultures at mRNA level. In pellet cultures of MSCs, TGF-β1 successfully induced chondrogenic differentiation and glycosaminoglycan synthesis. Addition of CNP into the TGF-β1 supplemented chondrogenic differentiation medium further induced the glycosaminoglycan synthesis and hypertrophy of differentiated chondrocytes in these pellets. Pellets induced with TGF-β1 and treated with antibodies against CNP and NPR-B, did show collagen type II expression, however, Alcian blue staining showing glycosaminoglycan synthesis was significantly suppressed. In conclusion, CNP-3/NPR-B signaling may strongly be involved in synthesis of glycosaminoglycans of the chondrogenic matrix and hypertrophy of differentiated chondrocytes during TGF-β1 induced chondrogenic differentiation of MSCs.     

Chondrogenesis of human periosteum-derived progenitor cells in atelocollagen

Periosteum-derived progenitor cells (PDPCs) could be differentiated into cartilage using atelocollagen as a carrier and in the presence of transforming growth factor-β3 (TGF-β3). Chondrogenesis was verified by RT-PCR and Western blotting. Expression of the type II collagen mRNA was found from the differentiated PDPCs in atelocollagen 3 weeks after chondrogenic induction. The chondrogenic potential of the PDPCs was also verified by histochemical staining for type II collagen protein. Increased production of glycosaminoglycan shows that the PDPCs in atelocollagen could differentiate into chondrocytes under a chondrogenic environment. PDPCs can therefore be used as a cell source for cell-based therapies targeted toward the articular cartilage of the knee.     

Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels: influence of collagen type II extracellular matrix on MSC chondrogenesis

Bone marrow mesenchymal stem cells (MSCs) are candidate cells for cartilage tissue engineering. This is due to their ability to undergo chondrogenic differentiation after extensive expansion in vitro and stimulation with various biomaterials in three-dimensional (3-D) systems. Collagen type II is one of the major components of the hyaline cartilage and plays a key role in maintaining chondrocyte function. This study aimed at analyzing the MSC chondrogenic response during culture in different types of extracellular matrix (ECM) with a focus on the influence of collagen type II on MSC chondrogenesis. Bovine MSCs were cultured in monolayer as well as in alginate and collagen type I and II hydrogels, in both serum free medium and medium supplemented with transforming growth factor (TGF) beta1. Chondrogenic differentiation was detected after 3 days of culture in 3-D hydrogels, by examining the presence of glycosaminoglycan and newly synthesized collagen type II in the ECM. Differentiation was most prominent in cells cultured in collagen type II hydrogel, and it increased in a time-dependent manner. The expression levels of the of chondrocyte specific genes: sox9, collagen type II, aggrecan, and COMP were measured by quantitative "Real Time" RT-PCR, and genes distribution in the hydrogel beads were localized by in situ hybridization. All genes were upregulated by the presence of collagen, particularly type II, in the ECM. Additionally, the chondrogenic influence of TGF beta1 on MSCs cultured in collagen-incorporated ECM was analyzed. TGF beta1 and dexamethasone treatment in the presence of collagen type II provided more favorable conditions for expression of the chondrogenic phenotype. In this study, we demonstrated that collagen type II alone has the potential to induce and maintain MSC chondrogenesis, and prior interaction with TGF beta1 to enhance the differentiation.     

Molecular and cellular characterization during chondrogenic differentiation of adipose tissue-derived stromal cells in vitro and cartilage formation in vivo

Human adipose tissue is a viable source of mesenchymal stem cells (MSCs) with wide differentiation potential for musculoskeletal tissue engineering research. The stem cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and expanded in vitro easily. This study was to determine molecular and cellular characterization of PLA cells during chondrogenic differentiation in vitro and cartilage formation in vivo . When cultured in vitro with chondrogenic medium as monolayers in high density, they could be induced toward the chondrogenic lineages. To determine their ability of cartilage formation in vivo , the induced cells in alginate gel were implanted in nude mice subcutaneously for up to 20 weeks. Histological and immunohistochemical analysis of the induced cells and retrieved specimens from nude mice at various intervals showed obviously cartilaginous phenotype with positive staining of specific extracellular matrix (ECM). Correlatively, results of RT-PCR and Western Blot confirmed the expression of characteristic molecules during chondrogenic differentiation namely collagen type II, SOX9, cartilage oligomeric protein (COMP) and the cartilage-specific proteoglycan aggrecan. Meanwhile, there was low level synthesis of collagen type X and decreasing production of collagen type I during induction in vitro and formation of cartilaginous tissue in vivo . These cells induced to form engineered cartilage can maintain the stable phenotype and indicate no sign of hypertrophy in 20 weeks in vivo , however, when they cultured as monolayers, they showed prehypertrophic alteration in late stage about 10 weeks after induction. Therefore, it is suggested that human adipose tissue may represent a novel plentiful source of multipotential stem cells capable of undergoing chondrogenesis and forming engineered cartilage.     

Chondrogenic differentiation of human mesenchymal stem cells cultured in a cobweb-like biodegradable scaffold

Human mesenchymal stem cells (MSCs) were cultured in vitro in a cobweb-like biodegradable polymer scaffold: a poly(dl-lactic-co-glycolic acid)-collagen hybrid mesh in serum-free DMEM containing TGF-beta3 for 1-10 weeks. The cells adhered to the hybrid mesh, distributed evenly, and proliferated to fill the spaces in the scaffold. The ability of the cells to express gene encoding type I collagen decreased, whereas its ability to express type II collagen and aggrecan increased. Histological examination by HE staining indicated that the cells showed fibroblast morphology at the early stage and became round after culture for 4 weeks. The cartilaginous matrices were positively stained by safranin O and toluidine blue. Immunostaining with anti-type II collagen and anti-cartilage proteoglycan showed that type II collagen and cartilage proteoglycan were detected around the cells. In addition, a homogeneous distribution of cartilaginous extracellular matrices was detected around the cells. These results suggest the chondrogenic differentiation of the mesenchymal stem cells in the hybrid mesh. The PLGA-collagen hybrid mesh enabled the aggregation of mesenchymal stem cells and provided a promotive microenvironment for the chondrogenic differentiation of the MSCs.     

The effective mode of growth and differentiation factor-5 in promoting the chondrogenic differentiation of adipose-derived stromal cells

Our study aimed to find out the most effective mode for chondrogenic differentiation based on time, dose and culture method. ADSCs were cultured and identified by CD44, CD49d, and CD106 immumohistochemical staining method, and their differentiation potential to chondrocyte were detected by Alizarin red staining. ADSCs induced by different concentrations of GDF-5 for chondrogenic differentiation were detected by blue and toluidine blue staining and collagen type II and X immumohistochemical staining. The expression of collagen I, II, X and aggrecan gene in GDF-induced ADSCs cultured in 2- and 3-dimension was identified by real-time PCR. Cell microstructure and proliferation in three-dimensional scaffolds at day 7, 14, 21 and 28 were analyzed by scanning electron microscopy and MTS assay. The ADSCs were successfully identified by CD44 and CD49d, and their differentiation potential was detected by Alizarin red staining. Real-time PCR showed that collagen and aggrecan were expressed at high levels in 100 or 200 ng/mL GDF-5 treated cells. The collagen types (I, II) and aggrecan genes were higher expressed in GDF-5 induced scaffold group than that in monolayer group. MTS showed that the cell counts were not significantly different among different treated time. Both collagen type II and aggrecan gene were highly expressed at day 14, while collagen types I and X gene expressions peaked at day 21 and 28. The 100 ng/mL GDF-5 is effective and cost-effective for chondrogenic differentiation when cultured at day 14 in vitro under three-dimensional culture conditions.     

Analysis of collagen expression during chondrogenic induction of human bone marrow mesenchymal stem cells

Adult mesenchymal stem cells (MSCs) are currently being investigated as an alternative to chondrocytes for repairing cartilage defects. As several collagen types participate in the formation of cartilage-specific extracellular matrix, we have investigated their gene expression levels during MSC chondrogenic induction. Bone marrow MSCs were cultured in pellet in the presence of BMP-2 and TGF-β3 for 24 days. After addition of FGF-2, at the fourth passage during MSC expansion, there was an enhancing effect on specific cartilage gene expression when compared to that without FGF-2 at day 12 in pellet culture. A switch in expression from the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of the collagen type II gene was observed at day 24. A short-term addition of FGF-2 followed by a treatment with BMP-2/TGF-β3 appears sufficient to accelerate chondrogenesis with a particular effect on the main cartilage collagens.     

Dynamic compressive strain influences chondrogenic gene expression in human mesenchymal stem cells

This study tests the hypothesis that dynamic compressive strain selectively enhances chondrogenic differentiation by human mesenchymal stem cells (MSCs). Primary MSCs were isolated and expended in monolayer culture. The cells were seeded in alginate constructs or in pellet culture. The time course of chondrogenic differentiation was assessed by real-time QPCR of mRNA expression analysis for cartilage specific markers. Collagen types II and X mRNA, not present in undifferentiated MSCs, were detectable by 2-4 days of chondrogenic induction and continued to rise significantly throughout the culture period of 10 days (p < 0.001). Basal levels of gene expression for Sox-9 and aggrecan were evident in undifferentiated MSCs, although chondrogenic induction for a period of 8 days resulted in an increased trend in the gene expression levels. The alginate system was also used in mechanical conditioning studies. Dynamic compression was applied, in an intermittent regimen, at a strain amplitude of 15% and frequency of 1 Hz in the presence and absence of 10 ng/ml TGFbeta3, for a period of 8 days. Results indicated significant changes in the levels of mRNA expression for the chondrogenic markers. For example, by day 8, the application of the strain regimen alone caused an up-regulation in all the chondrogenic markers compared to the control samples (no TGFbeta, no compression). However, the combined effects of strain and TGFbeta on these markers were more complex than purely additive.     

Regenerative Therapies for Equine Degenerative Joint Disease: A Preliminary Study

Degenerative joint disease (DJD) is a major cause of reduced athletic function and retirement in equine performers. For this reason, regenerative therapies for DJD have gained increasing interest. Platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) were isolated from a 6-year-old donor horse. MSCs were either used in their native state or after chondrogenic induction. In an initial study, 20 horses with naturally occurring DJD in the fetlock joint were divided in 4 groups and injected with the following: 1) PRP; 2) MSCs; 3) MSCs and PRP; or 4) chondrogenic induced MSCs and PRP. The horses were then evaluated by means of a clinical scoring system after 6 weeks (T₁), 12 weeks (T₂), 6 months (T₃) and 12 months (T₄) post injection. In a second study, 30 horses with the same medical background were randomly assigned to one of the two combination therapies and evaluated at T₁. The protein expression profile of native MSCs was found to be negative for major histocompatibility (MHC) II and p63, low in MHC I and positive for Ki67, collagen type II (Col II) and Vimentin. Chondrogenic induction resulted in increased mRNA expression of aggrecan, Col II and cartilage oligomeric matrix protein (COMP) as well as in increased protein expression of p63 and glycosaminoglycan, but in decreased protein expression of Ki67. The combined use of PRP and MSCs significantly improved the functionality and sustainability of damaged joints from 6 weeks until 12 months after treatment, compared to PRP treatment alone. The highest short-term clinical evolution scores were obtained with chondrogenic induced MSCs and PRP. This study reports successful in vitro chondrogenic induction of equine MSCs. In vivo application of (induced) MSCs together with PRP in horses suffering from DJD in the fetlock joint resulted in a significant clinical improvement until 12 months after treatment.     

Fibrin gel improved the spatial uniformity and phenotype of human chondrocytes seeded on collagen scaffolds

A scaffold made of equine collagen type I based material has been assessed for its use in the preparation of tissue-engineered cartilage implants with human articular chondrocytes. Improvements of cell-seeding efficiency and specific gene expression were studied by combining solid scaffold with fibrin glue or human blood plasma. Following 3 weeks of static culture, mRNA expression levels of collagen type I, collagen type II, aggrecan and versican were analyzed by real-time quantitative PCR and compared to those in native cartilage and monolayer cell cultures.Constructs prepared with fibrin glue or plasma showed higher cell seeding efficiencies than those prepared without gel. Chondrocytes seeded directly onto a collagen scaffold appeared fibroblastic in shape while those encapsulated in fibrin gel were spherical. The presence of fibrin glue positively influences on mRNA levels of collagen type II and aggrecan, while blood plasma enhanced only the level of collagen type II expression. Levels of collagen type I and versican decreased in presence of fibrin glue.In orthopaedics, the combination of solid collagen fleece with fibrin gel for implant preparation is seen to be preferred over solid material or even cells in a suspension, since fibrin gel improves seeding capacity of the scaffold, supports equal distribution of cells and stimulates higher chondrogenic phenotype expression.     

BMP-6 enhances chondrogenesis in a subpopulation of human marrow stromal cells

Marrow stromal cells (MSCs) can differentiate into several mesenchymal lineages. MSCs were recently shown to form cartilage in micromass cultures with serum-free medium containing TGF-beta and dexamethasone. Here we found that addition of BMP-6 increased the weight of the pellets about 10-fold and they stained more extensively for proteoglycans. mRNAs for type II procollagen and type X collagen were detected at 1 week and the levels were increased at 3 weeks. We also compared two subpopulation of cultures of MSCs: Small and rapidly self-renewing cells (RS cells) and the large, more mature and slowly replicating cells (mMSCs). The cartilage pellets prepared from cultures enriched for RS cells were about 2.5-fold larger, stained more extensively for proteoglycans, and had levels of mRNA for type II procollagen that were 1.6-fold higher. Also, RS cells retained more of their chondrogenic potential as the cells were passaged.     

Expression of the heparan sulfate proteoglycan, perlecan, during mouse embryogenesis and perlecan chondrogenic activity in vitro.

Expression of the basement membrane heparan sulfate proteoglycan (HSPG), perlecan (Pln), mRNA, and protein has been examined during murine development. Both Pln mRNA and protein are highly expressed in cartilaginous regions of developing mouse embryos, but not in areas of membranous bone formation. Initially detected at low levels in precartilaginous areas of d 12.5 embryos, Pln protein accumulates in these regions through d 15.5 at which time high levels are detected in the cartilage primordia. Laminin and collagen type IV, other basal lamina proteins commonly found colocalized with Pln, are absent from the cartilage primordia. Accumulation of Pln mRNA, detected by in situ hybridization, was increased in d 14.5 embryos. Cartilage primordia expression decreased to levels similar to that of the surrounding tissue at d 15.5. Pln accumulation in developing cartilage is preceded by that of collagen type II. To gain insight into Pln function in chondrogenesis, an assay was developed to assess the potential inductive activity of Pln using multipotential 10T1/2 murine embryonic fibroblast cells. Culture on Pln, but not on a variety of other matrices, stimulated extensive formation of dense nodules reminiscent of embryonic cartilaginous condensations. These nodules stained intensely with Alcian blue and collagen type II antibodies. mRNA encoding chondrocyte markers including collagen type II, aggrecan, and Pln was elevated in 10T1/2 cells cultured on Pln. Human chondrocytes that otherwise rapidly dedifferentiate during in vitro culture also formed nodules and expressed high levels of chondrocytic marker proteins when cultured on Pln. Collectively, these studies demonstrate that Pln is not only a marker of chondrogenesis, but also strongly potentiates chondrogenic differentiation in vitro.