Focal changes in blood supply during normal epiphyseal growth are central in the pathogenesis of osteochondrosis in pigs

Cartilage canals are temporary vessel-containing structures within the growth cartilage. The canals gradually regress with age in a process designated chondrification, where the content of the canals is replaced by cartilage. The process of chondrification is considered physiological; however, premature regression has been associated with the formation of lesions of osteochondrosis. The purpose of the present study was to gain further insight into the nature of and relationship between chondrification of cartilage canals and the initial steps in the pathogenesis of osteochondrosis with respect to morphology, presence of factors influencing the processes, and mode of cellular death. The articular–epiphyseal cartilage complexes of the distal femur of 48 pigs were studied, combining a technique of perfusion and tissue clearing with histological and immunohistological methods.

The results provided strong evidence that the process of chondrification occurs at the terminations of the cartilage canals and is characterized by disintegration of the endothelial cells and transformation of perivascular cells into matrix-producing chondrocytes. Lesions of osteochondrosis, however, are characterized by necrosis of the portion of the affected cartilage canal distal to a point of interruption, with subsequent necrosis of adjacent resting zone growth cartilage. This interruption of the cartilage canal blood supply occurs at the junction between cartilage and bone where anastomoses are formed between cartilage canal vessels and vessels from the bone marrow. It is possible that microfractures occurring secondary to minor trauma at a vulnerable time in the development of the cartilage may be the initial event in pathogenesis of osteochondrosis.     

Ischemic necrosis of cartilage in spontaneous and experimental lesions of osteochondrosis

This study was designed to examine the association of spontaneous lesions of osteochondrosis with vascular supply to epiphyseal cartilage, and to determine whether similar lesions could be experimentally reproduced by selective interruption of cartilage canal blood supply. The vascular supply to the articular-epiphyseal cartilage complex of the distal end of the femur was studied in 27 microfil- or barium-injected and cleared specimens and 24 serially sectioned microangiographic specimens from 27 clinically normal female swine (3.6 to 71.0 kg). Blood vessels supplying the articular-epiphyseal cartilage complex were consistently restricted to the epiphyseal region and the number of vessels decreased as the pigs increased in weight (p less than 0.001). Spontaneous lesions of osteochondrosis (i.e., cartilage necrosis) were initially seen in the first areas of epiphyseal cartilage to become avascular and were associated with necrotic blood vessels. The number and size of foci of necrotic cartilage increased as the pigs increased in weight (p less than 0.001). Blood supply to epiphyseal cartilage from cartilage canal vessels was surgically interrupted in a highly vascular area of the medial femoral condyle in eight additional 23-kg female swine. This procedure resulted in necrosis of blood vessels within cartilage canals followed by necrosis of surrounding cartilage, lesions that appeared to be identical to early spontaneous lesions of osteochondrosis. These results suggest that the viability of epiphyseal cartilage in the articular-epiphyseal cartilage complex is highly dependent on an adequate blood supply from cartilage canal vessels, and strongly implicates a defect in blood supply in the pathogenesis of osteochondrosis.     

Excessive degradation of type II collagen in articular cartilage in equine osteochondrosis.

Articular osteochondrosis (OCD) occurs in both man and animals. The etiology remains to be determined. Studies of OCD lesions in animals may provide clues as to its pathogenesis. The aim of our study was to determine whether there was evidence for increased degradation namely proteoglycan (PG) release and type II collagen cleavage in articular cartilage harvested from OCD lesions. We examined ex vivo explants at post-mortem from equine OCD lesions and macroscopically normal site and age matched cartilage. These were cultured over a 10 day period in serum-free medium. Type II collagen cleavage was measured in articular cartilage and media using an Elisa assay to detect the COL2-3/4C(short) epitope, which is generated on cleavage of the triple helix of type II collagen by collagenases. PG release was measured by a dye-binding assay. Cumulative release of PG and COL2-3/4C(short) and their contents in cartilage at the end of the culture period were determined. In OCD lesions there was a significant increase in type II collagen cleavage by collagenase but no evidence for increase of PG degradation. These findings point to a selective increase in type II collagen cleavage by collagenases, in OCD lesions of the kind observed in osteoarthritis. Further work is needed to determine whether changes represent primary or secondary events in the pathogenesis of OCD.     

The ultrastructure of osteochondrosis of the articular-epiphyseal cartilage complex in growing swine

Summary Osteochondrosis of the articular-epiphyseal cartilage complex (A-E complex) is a significant clinical disease in swine. It has been suggested that osteochondrosis is the underlying cause of osteochondritis dissecans in humans. The purpose of this investigation was to characterize the ultrastructural changes in the earliest macroscopically visible lesion of the epiphyseal cartilage in osteochondrosis of the A-E complex in swine. Osteochondritic epiphyseal cartilage from the distal femora and humeri of growing crossbred boars was collected, embedded in plastic, and studied light and electron microscopically. The predominant lesion was chondronecrosis, characterized by chondrocyte death and loss of matrical proteoglycan. Transition from normal to abnormal cartilage was abrupt. Lipid accumulated in chondrocytes within and adjacent to lesions, but not in chondrocytes distant from lesions. Intracellular lipid accumulation was an important feature of the lesion and may play a role in its initiation. It is hypothesized that intracellular lipid accumulation results from hypoxia/anoxia and may precede matrix degeneration, which precedes cell death.     

一氧化氮诱导腰椎软骨终板退变的实验研究

目的探讨一氧化氮（nitricoxide，NO）诱导腰椎软骨终板退变的机制。方法取长枫杂种猪腰椎间盘软骨终板细胞，于含20％胎牛血清的DMEM中培养，建立体外软骨终板细胞培养模型，以光镜及苏木精-伊红染色观察其生物学表现，Ⅱ型胶原免疫细胞化学染色对软骨终板细胞进行鉴定，以外源性NO-硝普钠（sodium nitroprusside，SNP）2mmol／l处理软骨终板细胞、^35S掺入法和^3H-脯氨酸掺入法检测蛋白多糖及胶原的合成，放射性免疫测定法测炎性细胞因子IL-1β、IL-6、TNF-α的生成，碘化丙啶荧光染色及DNA片段电泳观察软骨终板细胞凋亡。结果原代细胞生物学性状最接近体内细胞，多次传代后细胞呈现衰老现象，经免疫细胞化学染色证实此细胞具有Ⅱ型胶原表达。施加SNP后，软骨终板细胞蛋白多糖及胶原合成明显减少（P〈0．01），炎性细胞因子含量明显升高（P〈0．01），细胞凋亡率显著增高（P〈0．01）。结论NO抑制软骨终板细胞蛋白多糖及胶原合成，促进炎性细胞因子及细胞凋亡增加，从而促进软骨终板退变。     

Growth and integration of neocartilage with native cartilage in vitro.

Poor integration of neocartilage with recipient has been a major obstacle to articular cartilage restoration. An in vitro study was designed to provide insights regarding the integration process. Cartilage explants and chondrocytes were harvested from the distal sternum of 16-day-old chick embryos. Four million chondrocytes and one 1mm(3) explant were centrifuged together in a 0.75ml tube. In the constructs, consisting of cartilage explant and chondrocyte pellet, isolated chondrocytes attached to the surface of the explant at the beginning of the culture, followed by significant chondrocyte death at the interface between chondrocyte pellet and explant. Chondrocyte apoptosis was seen almost exclusively at this interface. Meanwhile, the interface was a zone with active extracellular matrix deposition as demonstrated by immunohistochemistry. By two weeks, the junction of neocartilage and native cartilage explant had formed an acellular zone with collagen fibrils orientated parallel with the surface of the cartilage explant. In conclusion, chondrocyte death leads to acellularity and fibril network reorganization at the neocartilage/explant interface, and impacts the quality of cartilage repair as abnormal matrix remodeling implies.     

Repair of porcine articular cartilage defect with autologous chondrocyte transplantation.

Articular cartilage is known to have poor healing capacity after injury. Autologous chondral grafting remains the mainstay to treat well-defined, full-thickness, symptomatic cartilage defects. We demonstrated the utilization of gelatin microbeads to deliver autologous chondrocytes for in vivo cartilage generation. Chondrocytes were harvested from the left forelimbs of 12 Lee-Sung pigs. The cells were expanded in monolayer culture and then seeded onto gelatin microbeads or left in monolayer. Shortly before implantation, the cell-laden beads were mixed with collagen type I gel, while the cells in monolayer culture were collected and re-suspended in culture medium. Full-thickness cartilage defects were surgically created in the weight-bearing surface of the femoral condyles of both knees, covered by periosteal patches taken from proximal tibia, and sealed with a porcine fibrin glue. In total, 48 condyles were equally allotted to experimental, control, and null groups that were filled beneath the patch with chondrocyte-laden beads in gel, chondrocytes in plain medium solution, or nothing, respectively. The repair was examined 6 months post-surgery on the basis of macroscopic appearance, histological scores based on the International Cartilage Repair Society Scale, and the proportion of characteristic chondrocytes. Tensile stress-relaxation behavior was determined from uniaxial indentation tests. The experimental group scored higher than the control group in the categories of matrix nature, cell distribution pattern, and absence of mineralization, with similar surface smoothness. Both the experimental and control groups were superior to the null group in the above-mentioned categories. Viable cell populations were equal in all groups, but the proportion of characteristic chondrocytes was highest in the experimental group. Matrix stiffness was ranked as null > native cartilage > control > experimental group. Transplanted autologous chondrocytes survive and could yield hyaline-like cartilage. The application of beads and gel for transplantation helped to retain the transferred cells in situ and maintain a better chondrocyte phenotype.     

Cellular turnover at the chondro-osseous junction of growth plate cartilage: analysis by serial sections at the light microscopical level

In the distal hypertrophic cell zone of growth plate cartilage, the penetration of metaphyseal vascular endothelial cells is into the noncalcified territorial and pericellular matrices. Cellular mechanisms that promote metaphyseal vascularization are understood poorly, partly because no study has addressed the question of the time sequence of cellular interactions at the chondro-osseous junction. The purpose of the present study is to make predictions about the relative and the real time duration of cellular events during vascular invasion, including an analysis of the time sequence of death of the terminal hypertrophic chondrocyte. The data from serial section analysis at the light microscopical level of tetracycline-labeled growth plates indicate that death of the terminal hypertrophic chondrocyte occurs in discrete morphological stages characterized by rapid cellular condensation followed, within minutes, by endothelial cell penetration into the vacated lacuna. Cellular condensation lasts approximately 45 min or 18% of the time a cell spends as a terminal chondrocyte. The data also demonstrate that chondrocytic death occurs prior to invasion by vascular endothelial cells and that the chondrocytic lacuna remains empty for as long as 15 min before an endothelial cell or blood vascular cell fills the space.     

永生化软骨细胞为基础的工程化软骨修复软骨缺损的实验研究

目的探讨用永生化软骨细胞作为种子细胞修复羊关节软骨缺损的可行性.方法把人端粒酶催化亚基(hTERT)转染羊关节软骨细胞,筛选阳性克隆并通过旋转生物反应器-微载体技术进行大量扩增;将扩增的永生化软骨细胞与β-磷酸三钙复合并在体外培育后,植入羊前肢肱骨头关节面软骨缺损处;3和6个月取材,进行形态学和免疫组织化学评价.结果实验组,术后6个月,缺损区被新生的软骨组织所充填,Ⅱ型胶原染色呈强阳性,与对照组差别有显著性差异(P＜0.01).在材料组,缺损区边缘可见部分新生软骨组织;空白对照组,缺损区新生软骨组织形成.结论永生化软骨细胞在软骨组织工程中具有潜在的应用前景.     

Canine osteoarthritis: Effects of endogenous neutral metalloproteoglycanases on articular cartilage proteoglycans

The aim of this study was to analyze the mechanisms by which neutral metalloproteoglycanases (NMPE) degrade proteoglycans (PGs) in the cartilage of an experimental model of osteoarthritis (OA). We demonstrated that chondrocytes in osteoarthritic cartilage synthesize PGs with the same functional characteristics as those found in normal cartilage. Osteoarthritic cartilage contains NMPE in both active and latent forms. Both forms can degrade newly synthesized and endogenous PG macromolecules, as indicated by the reduced hydrodynamic size found in the two PG populations of osteoarthritic cartilage. PG monomers, derived from the included fraction of Sepharose CL2B chromatography, were unable to form aggregates with hyaluronic acid. Reduction and alkylation showed that PG monomers from osteoarthritic cartilage had a small hydrodynamic size, especially after activation with aminophenylmercuric acetate. No significant differences were observed in the size of the chondroitin sulfate chain when normal cartilage was compared with its osteoarthritic equivalent. These results suggest that the proteolytic degradation of cartilage matrix PGs by NMPE occurs at both the hyaluronate-binding region and at the chondroitin sulfate-rich region of the core protein.     

Intercellular signaling as a cause of cell death in cyclically impacted cartilage explants

Recently, in vitro cartilage studies have shown that impact loading can produce structural damage and osteoarthritis-like changes, including tissue swelling, collagen denaturation, and cell death. OBJECTIVE: This study was to determine whether a signal for cell death moves through the cartilage matrix, resulting in the spread of cell death over time from impacted to unimpacted regions. DESIGN: Cyclic impacts were applied to the 2 mm core of 4 mm cartilage discs. Post-impact culturing extended for 3, 6 or 21 days and occurred in one of two ways. In one, discs were cultured intact. In the second, cores were removed immediately after cessation of impact and cores and rings cultured separately. Cells in apoptosis and later stage necrosis were monitored using the TUNEL assay. RESULTS: The extent of cell death in impacted samples increased with increased duration of post-impact culturing. At the early time, the majority of cell death was located in the regions of direct impact whereas after extended culture, the extent of cell death was similar in the surrounding unimpacted regions and in the impacted core region. However, the physical separation of the impacted core from the surrounding, non-impacted ring regions immediately after impact, and prior to independent culture, kept the level of cell death in the surrounding ring close to control levels, even after 21 days of incubation. DISCUSSION: These findings indicate that soluble intercellular signalling is involved in the spreading of cell death through the cartilage matrix, and that its effects can be prevented by physical isolation of the surrounding ring from the impacted core.     

Experimental verification of the role of interstitial fluid pressurization in cartilage lubrication.

The objective of the current study was to measure the friction coefficient simultaneously with the interstitial fluid load support in bovine articular cartilage, while sliding against glass under a constant load. Ten visually normal 6-mm-diameter cartilage plugs harvested from the humeral head of four bovine shoulder joints (ages 2-4 months) were tested in a custom friction device under reciprocating linear motion (range of translation +/-2 mm; sliding velocity 1 mm/s), subjected to a 4.5 N constant load. The frictional coefficient was found to increase with time from a minimum value of mu min=0.010+/-0.007 (mean+/-SD) to a maximum value of 0.243+/-0.044 over a duration ranging from 920 to 19,870 s (median: 4,560 s). The corresponding interstitial fluid load support decreased from a maximum of 88.8+/-3.8% to 8.7+/-8.6%. A linear correlation was observed between the frictional coefficient and interstitial fluid load support (r2=0.96+/-0.03). These results support the hypothesis that the temporal variation of the frictional coefficient correlates negatively with the interstitial fluid load support and that consequently interstitial fluid load support is a primary mechanism regulating the frictional response in articular cartilage. Fitting the experimental data to a previously proposed biphasic boundary lubrication model for cartilage yielded an equilibrium friction coefficient of mu eq=0.284+/-0.044. The fraction of the apparent contact area over which the solid cartilage matrix was in contact with the glass slide was predicted at phi s=1.7+/-6.3%, significantly smaller than the solid volume fraction of the tissue, phi s=13.8+/-1.8%. The model predictions suggest that mixed lubrication prevailed at the contact interface under the loading conditions employed in this study.     

Experimental evaluation of the usefulness of osteochondral allograft for articular cartilage defect

This study was conducted to evaluate the usefulness of osteochondral allografts for articular cartilage defects. Cartilaginous defects measuring 4.5mm in diameter were experimentally prepared in both the weight-bearing and non-weight-bearing regions of the femur in six male miniature pigs (9 months old). Osteochondral grafting was performed using fresh autografts (group AU), fresh allografts (group AL), or frozen allografts (group FA). Untreated cartilaginous defects were used as the control (group D). All the pigs were killed 4 weeks later, and the respective osteochondral grafts were macro- and microscopically evaluated. Hematoxylin and eosin staining, safranin O staining, and immunostaining [matrix metalloprotease-1 (MMP-1) and tissue inhibitor of metalloprotease-2 (TIMP-2)] were used for the histological evaluations of transplanted cartilage. Macroscopic and microscopic findings were assessed according to the criteria proposed by Wakitani et al. Although groups AU and AL showed similar median scores (ranges) for the evaluation of cartilaginous defect restoration, groups FA and D showed unfavorable scores: 3.9 (0–9) in group AU; 4.5 (0–12) in group AL; 10.2 (4–12) in group FA; and 7.0 (5–11) in group D. Immunostaining revealed almost identical results in groups AU and AL. As there were no histologically significant differences in the status of the osteochondral grafts between fresh autografts and fresh allografts, fresh allografts might be useful donor osteochondral grafts.     

大鼠脊髓三种不同损伤后神经细胞凋亡的实验比较

目的了解三种不同脊髓机械性损伤对继发性神经细胞凋亡的影响。方法采用脊髓挫伤、持续性占位、脊髓横断三种大鼠损伤模型,在伤后1、4、7d观察损伤断面神经细胞凋亡现象。结果脊髓挫伤组:灰质三个时间组的凋亡指数波动明显,灰质中凋亡指数4d时最高;白质中凋亡细胞除背侧束外腹侧束也存在;另外细胞凋亡以少突胶质细胞凋亡为主。脊髓持续性占位损伤组:灰质、白质细胞凋亡指数在4d和7d组中差异无显著性意义(P>0.05),白质凋亡细胞在受压的背侧束、外侧束为主。脊髓横断损伤组:灰质、白质在三个时间组中均有大量凋亡细胞。结论大鼠脊髓损伤后神经细胞虽然存在主动死亡(细胞凋亡)方式,但凋亡在时间和部位上与损伤类型、损伤部位、损伤程度有关。     

Biochemical, biomechanical and histological properties of osteoarthritic porcine knee cartilage: implications for osteochondral transplantation

Introduction Cartilage lesions of the knee joint are frequently observed during arthroscopy and when surgical intervention is required, osteochondral autograft procedures are an established method of treatment. Frequently lesions are located on the medial femoral condyle (MFC), and typical donor locations for osteochondral grafts include the medial and lateral patellar groove. This technique provides good results, even when the quality of cartilage transplanted from an osteoarthritic joint is doubtful. This study characterizes biological, biomechanical and histological properties of cartilage explants from the patellar groove harvested from osteoarthritic joints. Materials and methods Cylindrical cartilage explants were harvested from the arthritic areas of the MFC as well as normal appearing regions of the medial and lateral patellar groove from porcine joints revealing various grades of osteoarthritis. Matrix synthesis rates were determined, and explants were investigated by mechanical testing and histology. Results Articular cartilage obtained from the typical donor areas of the medial and lateral patellar groove provided constant enhanced material properties, matrix synthesis rates and histological appearance compared to samples from the arthritic lesions of the MFC, even in joints with end-stage osteoarthritis of the MFC. No significant difference was found between patellar groove cartilage samples harvested from joints with different stages of osteoarthritis. Conclusion Our findings demonstrate that healthy appearing cartilage from the patellar groove does not undergo significant alterations in material properties due to the arthritic milieu present in osteoarthritic joints. Accordingly these locations provide a source of functional tissue for transplant procedures even in joints with end-stage osteoarthritis.     

颈椎终板软骨细胞凋亡的检测及相关意义

目的检测颈椎终板软骨细胞的细胞凋亡指数,探讨其在椎间盘退变中可能的作用机制。方法颈椎间盘终板及髓核取自我院行颈椎前路手术的35例颈椎椎间盘退变患者（退变组）和19例颈椎外伤患者（外伤组）。光镜观察退变组和外伤组终板和髓核的细胞密度,TUNEL法检测两组终板软骨细胞和髓核细胞的细胞凋亡指数,咔唑分光光度法比较两组髓核蛋白多糖含量。结果退变组终板细胞密度较外伤组减少（P〈0.05）,TUNEL染色显示退变组终板细胞凋亡指数为（34.6±16.1）%,外伤组为（20.1±9.3）%,两组间比较差异有统计学意义（P〈0.05）。Pearson相关分析显示,颈椎终板TUNEL染色阳性细胞率与终板细胞密度、髓核蛋白多糖含量之间呈负相关（r=—0.805,P=0.001;r=—0.677,P=0.023）,与髓核TUNEL阳性细胞率之间呈正相关（r=0.758,P=0.003）。结论颈椎退变终板软骨细胞凋亡率较高,推测在椎间盘退变过程中可能发挥重要作用;软骨细胞凋亡可能与髓核细胞凋亡增加、终板细胞密度与髓核蛋白多糖含量降低密切相关。     

Cytochrome oxidase activities of various cartilage tissues during growth and development

As rabbits increase in size from 165 grams to 2,205 grams body weight, cytochrome oxidase activity of epiphyseal cartilage homogenates does not change in manometric and spectrophotometric enzyme assays, whereas the oxidase activities of xiphisternal and ear cartilage homogenates decline. Light absorption spectra of cartilage tissues were made for the first time, using strips of rabbit ear and pieces of chick embryo cartilage. Such spectra reveal characteristic absorption maxima for the terminal respiratory complex of mitochondria, including b, c and oxidase components and flavoprotein.

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Zusammenfassung Während Kaninchen ihr Körpergewicht von 165 auf 2205 g erhöhen, erfolgt keine Veränderung in der Cytochrom-Oxydase-Aktivität der epiphysären Knorpel-Homogenate in manometrischen und spectrophotometrischen Enzymversuchen, jedoch vermindern sich die Oxydase-Aktivitäten von xiphisternalen und von Ohrknorpel-Homogenaten. Lichtabsorptionsspektren von Knorpelgewebe wurden zum ersten Mal gemacht, indem Streifen von Kaninchenohrstücken und Stücke von Kükenembryoknorpel verwendet wurden. Solche Spektren zeigen charakteristische Absorptionsmaxima für den terminalen Atmungskomplex der Mitochondrien, einschließlich b, c und Oxydasekomponenten sowie Flavoprotein.

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Résumé Lorsque des lapins augmentent pondéralement de 165 à 2,205 grammes, l'activité en cytochrome oxydase de cartilage épiphysaire homogénéisé n'est pas modifiée, en utilisant des tests enzymatiques manométriques et spectrophotométriques; alors que l'activité en oxydase du cartilage xyphoide du sternum et de l'oreille homogénéisé décroit. Des spectres d'absorption optique des tissus cartilagineux, réalisés pour la première fois à l'aide de fragments d'oreilles de lapin et de cartilage embryonnaire de poulets, montrent un maximum d'absorption caractéristique pour le système respiratoire final des mitochondries, y compris les composés b, c et oxydasiques ainsi que les flavoprotéines.

     

关节软骨缺损修复的实验与临床

关节软骨的修复一直是骨科领域尚未完全解决的一大难题。现就关节软骨损伤后促进自身修复、组织或细胞移植修复、组织工程修复等方面对关节软骨修复方法作一综述。