膜性高分子材料在兔眼部的生物相容性

目的评价聚羟基脂肪酸(polyhydroxyalkanoates,PHA)、聚乳酸(polylacetic acid,PLA)和聚己内酯(polycaprolactone,PCL)三种膜性高分子材料在兔眼部的生物相容性。方法将24只新西兰兔随机分为4组,每组6只。PHA、PLA、PCL为实验组,材料植入兔右眼结膜下。假手术组结膜下钝性分离,但不植入任何高分子材料。使用裂隙灯显微镜观察并记录植入后不同时间手术眼的反应并评分。裂隙灯下观察材料的吸收时间。术后4周和16周取眼球,行HE染色、Masson染色和天狼猩红染色分别定性观察组织结构和炎症细胞、胶原纤维和胶原纤维的亚型与排列方向。结果术眼刺激性评分等级各组均不高于"轻度刺激性"。结膜下吸收时间PHA、PLA和PCL组分别是16周,12周和大于16周。组织学观察术后4周PHA、PLA和PCL组均形成材料包裹囊腔,囊壁以纤维组织为主,伴有毛细血管形成和炎性细胞浸润,以中性粒细胞为主。胶原纤维染色与假手术组无明显差异,以Ⅰ型和Ⅲ型为主,大致呈平行排列。术后16周PHA和PLA组材料已不可查及,包裹囊腔结构不规则,而PCL材料整体可查及,包裹囊腔规则。各组未见毛细血管,偶见淋巴细胞浸润。胶原纤维与假手术组无明显差异,以Ⅰ型和Ⅲ型为主,仍大致呈平行排列。结论 PHA、PLA和PCL三种膜性高分子材料在兔眼具有较好的生物相容性,结膜下吸收时间分别是16周,12周和大于16周。     

The restoration of full-thickness cartilage defects with mesenchymal stem cells (MSCs) loaded and cross-linked bilayer collagen scaffolds on rabbit model

Cartilage has a limited self-repair capability and the repair of large cartilage defects remains a challenge in clinic. This study aimed to investigate the effect of mesenchymal stem cells (MSCs) loaded three-dimensional bilayer collagen scaffold for cartilage repair. Cross-linked three-dimensional bilayer collagen scaffolds seeded with or without MSCs were implanted into large cartilage defects (4 mm in diameter; 3 mm in depth) in rabbit knees. The untreated cartilage defects served as control. The tissue response was evaluated at 6 and 12 weeks after implantation by general histology and semi-quantitative histological grading systems. In addition, the repaired tissues were evaluated by mechanical test at 12 weeks after implantation. The MSCs-loaded collagen scaffold group showed the most hyaline cartilage, highest histological scores and compressive modulus. Moreover, it showed a good integration with the subchondral bone and adjacent cartilage. The structure of the novel bilayer collagen scaffolds provided architectural support for the differentiation of MSCs and demonstrated successful induction of in vivo chondrogenesis. These findings suggested that MSCs-loaded bilayer collagen scaffold could be an appealing candidate to be used for cartilage regeneration.     

聚乳酸聚乙醇酸膜、聚β羟基丁酯膜和胶原膜三种材料的相容性探讨

目的 评价聚乳酸聚乙醇酸膜、聚β羟基丁酯膜和胶原膜的结构、生物相容性及其在组织工程血管中的应用前景.方法 HE、胶原染色,扫描电镜观察材料的结构.新西兰兔15只皮下植入材料,于4、6、8和12周取出观察其组织反应和降解情况.将犬股动脉间质细胞种植于聚乳酸聚乙醇酸膜、胶原膜上,观察其形态.结果 聚乳酸聚乙醇酸膜、聚β羟基...     

The Graft of Autologous Adipose-Derived Stem Cells in the Corneal Stromal after Mechanic Damage

This study was designed to explore the feasibility of using autologous rabbit adipose derived stem cells (rASCs) as seed cells and polylactic-co-glycolic acid (PLGA) as a scaffold for repairing corneal stromal defects. rASCs isolated from rabbit nape adipose tissue were expanded and seeded on a PLGA scaffold to fabricate cell-scaffold constructs. After 1 week of cultivation in vitro, the cell-scaffold complexes were transplanted into corneal stromal defects in rabbits. In vivo, the autologous rASCs-PLGA constructed corneal stroma gradually became transparent without corneal neovascularization after 12 weeks. Hematoxylin and eosin staining and transmission electron microscopy examination revealed that their histological structure and collagen fibril distribution at 24 weeks after implantation were similar to native counterparts. As to the defect treated with PLGA alone, the stromal defects remained. And scar tissue was observed in the untreated-group. The implanted autologous ASCs survived up to 24 weeks post-transplantation and differentiated into functional keratocytes, as assessed by the expression of aldehyde-3-dehydrogenase1A1 (ALDH1A1) and cornea-specific proteoglycan keratocan. Our results revealed that autologous rASCs could be one of the cell sources for corneal stromal restoration in diseased corneas or for tissue engineering of a corneal equivalent.     

Biocompatibility Studies of a New Biosynthetic Dermal Substitute Based on Collagen/Hyaluronan Conjugate

The use of glycosaminoglycans (GAGs) as the second macromolecular component with collagen for the membrane preparation is based on the idea to create a biodegradable scaffold for new tissue. A hybrid collagen/hyaluronan membrane with specific bubble macrostructure was designed to serve as a synthetic dermal substitute. The objectives were to explain the two questions: what is the local biological response to the implanted membrane, and what is its reconstruction rate in tissue. The histological study proved low irritability, good compatibility, ingrowth of autologous tissue starting on day 7 post implantation, and resorption within four weeks. The major part of the implant was replaced by autologous tissue at the end of three weeks post implantation. The overall local biological response to implant was very good.     

Investigating the effects of preinduction on human adipose-derived precursor cells in an athymic rat model

The osteogenic potential of human adipose-derived precursor cells seeded on medical-grade polycaprolactone-tricalcium phosphate scaffolds was investigated in this in vivo study. Three study groups were investigated: (1) induced--stimulated with osteogenic factors only after seeding into scaffold; (2) preinduced--induced for 2 weeks before seeding into scaffolds; and (3) uninduced--cells without any introduced induction. For all groups, scaffolds were implanted subcutaneously into the dorsum of athymic rats. The scaffold/cell constructs were harvested at the end of 6 or 12 weeks and analyzed for osteogenesis. Gross morphological examination using scanning electron microscopy indicated good integration of host tissue with scaffold/cell constructs and extensive tissue infiltration into the scaffold interior. Alizarin Red histology and immunostaining showed a heightened level of mineralization and an increase in osteonectin, osteopontin, and collagen type I protein expression in both the induced and preinduced groups compared with the uninduced groups. However, no significant differences were observed in these indicators when compared between the induced and preinduced groups.     

Characteristics of cartilage engineered from human pediatric auricular cartilage

In the repair of cartilage defects, autologous tissue offers the advantage of lasting biocompatibility. The ability of bovine chondrocytes isolated from hyaline cartilage to generate tissue-engineered cartilage in a predetermined shape, such as a human ear, has been demonstrated; however, the potential of chondrocytes isolated from human elastic cartilage remains unknown. In this study, the authors examined the multiplication characteristics of human auricular chondrocytes and the ability of these cells to generate new elastic cartilage as a function of the length of time they are maintained in vitro. Human auricular cartilage, harvested from patients 5 to 17 years of age, was digested in collagenase, and the chondrocytes were isolated and cultured in vitro for up to 12 weeks. Cells were trypsinized, counted, and passaged every 2 weeks. Chondrocyte-polymer (polyglycolic acid) constructs were created at each passage and then implanted into athymic mice for 8 weeks. The ability of the cells to multiply in vitro and their ability to generate new cartilage as a function of the time they had been maintained in vitro were studied. A total of 31 experimental constructs from 12 patients were implanted and compared with a control group of constructs without chondrocytes. In parallel, a representative sample of cells was evaluated to determine the presence of collagen. The doubling rate of human auricular chondrocytes in vitro remained constant within the population studied. New tissue developed in 22 of 31 experimental implants. This tissue demonstrated the physical characteristics of auricular cartilage on gross inspection. Histologically, specimens exhibited dense cellularity and lacunae-containing cells embedded in a basophilic matrix. The specimens resembled immature cartilage and were partially devoid of the synthetic material of which the construct had been composed. Analyses for collagen, proteoglycans, and elastin were consistent with elastic cartilage. No cartilage was detected in the control implants. Human auricular chondrocytes multiply well in vitro and possess the ability to form new cartilage when seeded onto a three-dimensional scaffold. These growth characteristics might some day enable chondrocytes isolated from a small auricular biopsy to be expanded in vitro to generate a large, custom-shaped, autologous graft for clinical reconstruction of a cartilage defect, such as for congenital microtia.     

Transdermal photopolymerization of poly(ethylene oxide)-based injectable hydrogels for tissue-engineered cartilage

Transdermal photopolymerization, a minimally invasive method for implantation, was used to subcutaneously place a mixture of polymer and isolated chondrocytes to regenerate cartilage tissue in vivo. Semi-interpenetrating networks of varying proportions of poly(ethylene oxide)-dimethacrylate and poly(ethylene oxide) and primary bovine articular chondrocytes were implanted in athymic mice. Four mice (12 implants) were harvested at 2, 4, and 7 weeks. Chondrocytes survived implantation and photopolymerization and formed neocartilage containing 1.5 to 2.9% wet weight collagen and 4 to 7% glycosaminoglycan. Thirty-five percent of the total collagen was type II collagen. Histologic analysis exhibited tissue structure resembling neocartilage, and safranin O staining demonstrated glycosaminoglycan distribution throughout the hydrogels. This study demonstrates the potential use of transdermal photopolymerization for minimally invasive subcutaneous implantation of hydrogels and chondrocytes for in vivo cartilage regeneration.     

Three Dimensional Collagen Scaffold Promotes Intrinsic Vascularisation for Tissue Engineering Applications

Here, we describe a porous 3-dimensional collagen scaffold material that supports capillary formation in vitro, and promotes vascularization when implanted in vivo. Collagen scaffolds were synthesized from type I bovine collagen and have a uniform pore size of 80 μm. In vitro, scaffolds seeded with primary human microvascular endothelial cells suspended in human fibrin gel formed CD31 positive capillary-like structures with clear lumens. In vivo, after subcutaneous implantation in mice, cell-free collagen scaffolds were vascularized by host neovessels, whilst a gradual degradation of the scaffold material occurred over 8 weeks. Collagen scaffolds, impregnated with human fibrinogen gel, were implanted subcutaneously inside a chamber enclosing the femoral vessels in rats. Angiogenic sprouts from the femoral vessels invaded throughout the scaffolds and these degraded completely after 4 weeks. Vascular volume of the resulting constructs was greater than the vascular volume of constructs from chambers implanted with fibrinogen gel alone (42.7±5.0 μL in collagen scaffold vs 22.5±2.3 μL in fibrinogen gel alone; p<0.05, n = 7). In the same model, collagen scaffolds seeded with human adipose-derived stem cells (ASCs) produced greater increases in vascular volume than did cell-free collagen scaffolds (42.9±4.0 μL in collagen scaffold with human ASCs vs 25.7±1.9 μL in collagen scaffold alone; p<0.05, n = 4). In summary, these collagen scaffolds are biocompatible and could be used to grow more robust vascularized tissue engineering grafts with improved the survival of implanted cells. Such scaffolds could also be used as an assay model for studies on angiogenesis, 3-dimensional cell culture, and delivery of growth factors and cells in vivo.     

Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo.

Articular cartilage exhibits little intrinsic repair capacity, and new tissue engineering approaches are being developed to promote cartilage regeneration using cellular therapies. The goal of this study was to examine the chondrogenic potential of adipose tissue-derived stromal cells. Stromal cells were isolated from human subcutaneous adipose tissue obtained by liposuction and were expanded and grown in vitro with or without chondrogenic media in alginate culture. Adipose-derived stromal cells abundantly synthesized cartilage matrix molecules including collagen type II, VI, and chondroitin 4-sulfate. Alginate cell constructs grown in chondrogenic media for 2 weeks in vitro were then implanted subcutaneously in nude mice for 4 and 12 weeks. Immunohistochemical analysis of these samples showed significant production of cartilage matrix molecules. These findings document the ability of adipose tissue-derived stromal cells to produce characteristic cartilage matrix molecules in both in vitro and in vivo models, and suggest the potential of these cells in cartilage tissue engineering.     

Fates and osteogenic differentiation potential of human mesenchymal stem cells in immunocompromised mice

Human mesenchymal stem cells (hMSCs) from bone marrow were genetically marked by using a murine leukaemia virus construct encoding enhanced green fluorescent protein (eGFP). The marked cells were either directly implanted into the tibialis anterior muscle or introduced into a variety of other tissue sites in immunocompromised mice (NOD/SCID and C.B-17 SCID/beige) to investigate their fates and differentiation potentials. It was observed that the hMSCs survived for up to 12 weeks and showed site-specific morphological phenotypes. hMSCs delivered by intravenous injection were found mainly in the lungs and were detected rarely in other organs. Histomorphometry showed that, after implantation of hMSCs into the tibialis anterior muscle juxtaskeletally, the areas of reactive host callus formation at 1 and 2 weeks and of ectopic human bone formation at 1 week were significantly increased compared with the control group. Expression of eGFP and human RUNX2, alkaline phosphatase, osteocalcin, osteopontin, and collagen type I mRNAs were detected in mice implanted with the labelled hMSCs but not in sham-treated samples. Active clearance of the reactive callus and ectopic calcified tissue by osteoclast-like tartrate-resistant acid phosphatase-positive cells was observed. We conclude that the eGFP-labelled hMSCs can survive and retain the potential to differentiate morphologically into a variety of apparent mesenchymal phenotypes in vivo. Absolute confirmation of differentiation capacity requires further study and is complicated by known possibilities of fusion of donor and host cells or limited transfer of genetic material. Nevertheless, the genetically marked hMSCs are shown to participate extensively in bone formation and turnover. Control of the host osteoclast/macrophage responses resulting in clearance of formed osteogenic tissue warrants further investigation to promote prolonged human osteogenesis in immunocompromised mice. Furthermore, any proposed general cytotherapeutic strategy for enhanced osteogenesis is likely to require supplementation of local bone-forming biological signals.     

Mathematical modelling of implant in an operated hernia for estimation of the repair persistence

This paper presents mathematical modelling of an implanted surgical mesh used in the repair process of the abdominal hernia. The synthetic implant is simulated by a membrane structure. The author provides a material modelling of the implant based on the dense net model appropriate for technical fabrics. The accuracy of the proposed solution is evaluated by comparing the simulations of the dynamic behaviour of the system with the experiments carried out on physical models of implanted mesh. The model can be used to estimate the repair persistence for different mesh materials, fixing systems and different numbers of tacks to be provided during the surgery in order to resist the cough pressure and required action to avoid hernia recurrence. The persistence of the repaired hernia is assessed on the basis of the values of the forces in the tissue–implant joints because the usual form of the repair failure is due to as the joint disconnection or tissue failure.     

国产多孔钽材料修复兔胫骨缺损的实验研究

摘要 目的：研究国产多孔钽材料能否在兔胫骨缺损模型中顺利实现骨长入,用于修复胫骨缺损。方法：在36只新西兰大白兔双侧胫骨骨干处建立骨缺损模型,每只动物左右侧缺损随机分组,分别进入实验组(植入多孔坦材料)和对照组（不植入多孔坦材料）。植入后4周、8周和12周取材,通过X线检测以及硬组织切片苏木精伊红染色,检测多孔钽材料与骨界面的骨整合情况。采用推出实验检测多孔钽材料与骨界面的结合强度。结果：将术后不同时间点取得的胫骨标本作X射线拍片分析,4周时,骨缺损端与材料结合部位有骨质生成,在8周时材料表面有骨形成现象,逐渐完全覆盖材料表面,在12周时骨量继续增加,形成覆盖材料并桥接骨缺损断端的骨痂。样本行硬组织切片并行HE染色后检测,植入4周后实验组材料两端被新生骨所覆盖,材料深部的孔隙中也可见少量骨组织长入;植入8周后发现实验组材料与骨组织生长良好,多孔钽材料表面和两端材料孔隙内均有骨组织长入,材料孔隙与组织紧密连接,有骨小梁长入;植入12周时两端骨组织长入深度没有明显变化,但材料表面骨组织继续长入,并完全嵌入圆柱体材料内。材料植入后4周与8周比较差异无统计学意义（P＞0.05）,材料植入后8周与12周比较差异有统计学意义（P<0.05）。将植入4周、8周和12周后含材料样本置于动态疲劳试验机上进行推出实验,随时间延长所需推出力明显增加,植入后4周和8周相比,虽然后者所需推力较大,但两者比较差异无统计学意义（P＞0.05）,而8周和12周比较则差异有统计学意义（P<0.05）。结论：国产多孔坦材料能在胫骨缺损中实现与骨整合,能用于皮质骨缺损修复。     

Stem cell‐mediated natural tissue engineering

Recently, we demonstrated that a fully differentiated tissue developed on a ventricular septal occluder that had been implanted due to infarct‐related septum rupture. We suggested that this tissue originated from circulating stem cells. The aim of the present study was to evaluate this hypothesis and to investigate the physiological differentiation and transdifferentiation potential of circulating stem cells. We developed an animal model in which a freely floating membrane was inserted into each the left ventricle and the descending aorta. Membranes were removed after pre‐specified intervals of 3 days, and 2, 6 and 12 weeks; the newly developed tissue was evaluated using quantitative RT‐PCR, immunohistochemistry and in situ hybridization. The contribution of stem cells was directly evaluated in another group of animals that were by treated with granulocyte macrophage colony‐stimulating factor (GM‐CSF) early after implantation. We demonstrated the time‐dependent generation of a fully differentiated tissue composed of fibroblasts, myofibroblasts, smooth muscle cells, endothelial cells and new blood vessels. Cells differentiated into early cardiomyocytes on membranes implanted in the left ventricles but not on those implanted in the aortas. Stem cell mobilization with GM‐CSF led to more rapid tissue growth and differentiation. The GM‐CSF effect on cell proliferation outlasted the treat ment period by several weeks. Circulating stem cells contributed to the development of a fully differentiated tissue on membranes placed within the left ventricle or descending aorta under physiological conditions. Early cardiomyocyte generation was identified only on membranes positioned within the left ventricle.     

Novel chitosan/collagen scaffold containing transforming growth factor-beta1 DNA for periodontal tissue engineering

The current rapid progression in tissue engineering and local gene delivery system has enhanced our applications to periodontal tissue engineering. In this study, porous chitosan/collagen scaffolds were prepared through a freeze-drying process, and loaded with plasmid and adenoviral vector encoding human transforming growth factor-beta1 (TGF-beta1). These scaffolds were evaluated in vitro by analysis of microscopic structure, porosity, and cytocompatibility. Human periodontal ligament cells (HPLCs) were seeded in this scaffold, and gene transfection could be traced by green fluorescent protein (GFP). The expression of type I and type III collagen was detected with RT-PCR, and then these scaffolds were implanted subcutaneously into athymic mice. Results indicated that the pore diameter of the gene-combined scaffolds was lower than that of pure chitosan/collagen scaffold. The scaffold containing Ad-TGF-beta1 exhibited the highest proliferation rate, and the expression of type I and type III collagen up-regulated in Ad-TGF-beta1 scaffold. After implanted in vivo, EGFP-transfected HPLCs not only proliferated but also recruited surrounding tissue to grow in the scaffold. This study demonstrated the potential of chitosan/collagen scaffold combined Ad-TGF-beta1 as a good substrate candidate in periodontal tissue engineering.     

BMP-12 treatment of adult mesenchymal stem cells in vitro augments tendon-like tissue formation and defect repair in vivo

We characterized the differentiation of rat bone marrow-derived mesenchymal stem cells (BM-MSCs) into tenocyte-like cells in response to bone morphogenetic protein-12 (BMP-12). BM-MSCs were prepared from Sprague-Dawley rats and cultured as monolayers. Recombinant BMP-12 treatment (10 ng/ml) of BM-MSCs for 12 hours in vitro markedly increased expression of the tenocyte lineage markers scleraxis (Scx) and tenomodulin (Tnmd) over 14 days. Treatment with BMP-12 for a further 12-hour period had no additional effect. Colony formation assays revealed that ~80% of treated cells and their progeny were Scx- and Tnmd-positive. BM-MSCs seeded in collagen scaffolds and similarly treated with a single dose of BMP-12 also expressed high levels of Scx and Tnmd, as well as type I collagen and tenascin-c. Furthermore, when the treated BM-MSC-seeded scaffolds were implanted into surgically created tendon defects in vivo, robust formation of tendon-like tissue was observed after 21 days as evidenced by increased cell number, elongation and alignment along the tensile axis, greater matrix deposition and the elevated expression of tendon markers. These results indicate that brief stimulation with BMP-12 in vitro is sufficient to induce BM-MSC differentiation into tenocytes, and that this phenotype is sustained in vivo. This strategy of pretreating BM-MSCs with BMP-12 prior to in vivo transplantation may be useful in MSC-based tendon reconstruction or tissue engineering.     

SYNTHESIS, MIGRATION, AND RELEASE OF PRECURSOR COLLAGEN BY ODONTOBLASTS AS VISUALIZED BY RADIOAUTOGRAPHY AFTER [3H]PROLINE ADMINISTRATION

The elaboration of dentin collagen precursors by the odontoblasts in the incisor teeth of 30–40-g rats was investigated by electron microscopy, histochemistry, and radioautography after intravenous injection of tritium-labeled proline. At 2 min after injection, when the labeling of blood proline was high, radioactivity was restricted to the rough endoplasmic reticulum, indicating that it is the site of synthesis of the polypeptide precursors of collagen, the pro-alpha chains. At 10 min, when the labeling of blood proline had already declined, radioactivity was observed in spherical portions of Golgi saccules containing entangled threads, and, at 20 min, radioactivity appeared in cylindrical portions containing aggregates of parallel threads. The parallel threads measured 280–350 nm in length and stained with the low pH-phosphotungstic acid technique for carbohydrate and with the silver methenamine technique for aldehydes (as did extracellular collagen fibrils). The passage of label from spherical to cylindrical Golgi portions is associated with the reorganization of entangled into parallel threads, which is interpreted as the packing of procollagen molecules. Between 20 and 30 min, prosecretory and secretory granules respectively became labeled. These results indicate that the cylindrical portions of Golgi saccules transform into prosecretory and subsequently into secretory granules. Within these granules, the parallel threads, believed to be procollagen molecules, are transported to the odontoblast process. At 90 min and 4 h after injection, label was present in predentin, indicating that the labeled content of secretory granules had been released into predentin. This occurred by exocytosis as evidenced by the presence of secretory granules in fusion with the plasmalemma of the odontoblast process. It is proposed that pro-alpha chains give rise to procollagen molecules which assemble into parallel aggregates in the Golgi apparatus. Procollagen molecules are then transported within secretory granules to the odontoblast process and released by exocytosis. In predentin procollagen molecules would give rise to tropocollagen molecules, which would then polymerize into collagen fibrils.     

Effects of introducing cultured human chondrocytes into a human articular cartilage explant model

Articular cartilage (AC) heals poorly and effective host-tissue integration after reconstruction is a concern. We have investigated the ability of implanted chondrocytes to attach at the site of injury and to be incorporated into the decellularized host matrix adjacent to a defect in an in vitro human explant model. Human osteochondral dowels received a standardized injury, were seeded with passage 3 chondrocytes labelled with PKH 26 and compared with two control groups. All dowels were cultured in vitro, harvested at 0, 7, 14 and 28 days and assessed for chondrocyte adherence and migration into the region of decellularized tissue adjacent to the defects. Additional evaluation included cell viability, general morphology and collagen II production. Seeded chondrocytes adhered to the standardized defect and areas of lamina splendens disruption but did not migrate into the adjacent acellular region. A difference was noted in viable-cell density between the experimental group and one control group. A thin lattice-like network of matrix surrounded the seeded chondrocytes and collagen II was present. The results indicate that cultured human chondrocytes do indeed adhere to regions of AC matrix injury but do not migrate into the host tissue, despite the presence of viable cells. This human explant model is thus an effective tool for studying the interaction of implanted cells and host tissue.     

Early and stable upregulation of collagen type II,collagen type I and YKL40 expression levels in cartilage during early experimental osteoarthritis occurs independent of joint location and histological grading

While morphologic and biochemical aspects of degenerative joint disease (osteoarthritis [OA]) have been elucidated by numerous studies, the molecular mechanisms underlying the progressive loss of articular cartilage during OA development remain largely unknown. The main focus of the present study was to gain more insight into molecular changes during the very early stages of mechanically induced cartilage degeneration and to relate molecular alterations to histological changes at distinct localizations of the joint. Studies on human articular cartilage are hampered by the difficulty of obtaining normal tissue and early-stage OA tissue, and they allow no progressive follow-up. An experimental OA model in dogs with a slow natural history of OA (Pond–Nuki model) was therefore chosen. Anterior cruciate ligament transection (ACLT) was performed on 24 skeletally mature dogs to induce joint instability resulting in OA. Samples were taken from different joint areas after 6, 12, 24 and 48 weeks, and gene expression levels of common cartilage molecules were quantified in relation to the histological grading (modified Mankin score) of adjacent tissue. Histological changes reflected early progressive degenerative OA. Soon after ACLT, chondrocytes responded to the altered mechanical conditions by significant and stable elevation of collagen type II, collagen type I and YKL40 expression, which persisted throughout the study. In contrast to the mild to moderate histological alterations, these molecular changes were not progressive and were independent of the joint localization (tibia, femur, lateral, medial) and the extent of matrix degeneration. MMP13 remained unaltered until 24 weeks, and aggrecan and tenascinC remained unaltered until 48 weeks after ACLT. These findings indicate that elevated collagen type II, collagen type I and YKL40 mRNA expression levels are early and sensitive measures of ACLT-induced joint instability independent of a certain grade of morphological cartilage degeneration. A second phase of molecular changes in OA may begin around 48 weeks after ACLT with altered expression of further genes, such as MMP13, aggrecan and tenascin. Molecular changes observed in the present study suggest that dog cartilage responds to degenerative conditions by regulating the same genes in a similar direction as that observed for chondrocytes in late human OA.     

Creating an Animal Model of Tendinopathy by Inducing Chondrogenic Differentiation with Kartogenin

Previous animal studies have shown that long term rat treadmill running induces over-use tendinopathy, which manifests as proteoglycan accumulation and chondrocytes-like cells within the affected tendons. Creating this animal model of tendinopathy by long term treadmill running is however time-consuming, costly and may vary among animals. In this study, we used a new approach to develop an animal model of tendinopathy using kartogenin (KGN), a bio-compound that can stimulate endogenous stem/progenitor cells to differentiate into chondrocytes. KGN-beads were fabricated and implanted into rat Achilles tendons. Five weeks after implantation, chondrocytes and proteoglycan accumulation were found at the KGN implanted site. Vascularity as well as disorganization in collagen fibers were also present in the same site along with increased expression of the chondrocyte specific marker, collagen type II (Col. II). In vitro studies confirmed that KGN was released continuously from KGN-alginate in vivo beads and induced chondrogenic differentiation of tendon stem/progenitor cells (TSCs) suggesting that chondrogenesis after KGN-bead implantation into the rat tendons is likely due to the aberrant differentiation of TSCs into chondrocytes. Taken together, our results showed that KGN-alginate beads can be used to create a rat model of tendinopathy, which, at least in part, reproduces the features of over-use tendinopathy model created by long term treadmill running. This model is mechanistic (stem cell differentiation), highly reproducible and precise in creating localized tendinopathic lesions. It is expected that this model will be useful to evaluate the effects of various topical treatments such as NSAIDs and platelet-rich plasma (PRP) for the treatment of tendinopathy.