Integrative cartilage repair: adhesive strength is correlated with collagen deposition.

Procedures to repair focal articular cartilage defects often result in poor integration between the host cartilage and the graft tissue, and this may be related to the lack of matrix deposition and the death of chondrocytes near a cut cartilage surface. The objective of this study was to determine if cartilage repair was related to deposition of newly synthesized collagen. The mechanical integration that occurred between two live adult bovine cartilage blocks cultured in partial apposition for two weeks was correlated with [3H]proline incorporation, a measure of protein synthesis, of which more than 66% was accounted for by collagen. A similar level of mechanical integration occurred in sample pairs consisting of a live and killed cartilage block, and this adhesive strength was also correlated with [3H]proline deposition into both the live and the killed blocks. In these samples, the [3H]proline deposited into the killed cartilage appeared to originate from chondrocytes in the live cartilage, since live cells were not detected in the killed cartilage block by either viability staining or [35S]sulfate incorporation. These results suggest a mechanism of integrative cartilage repair in which live chondrocytes within cartilage secrete matrix molecules that are components of a collagen network, and subsequent deposition of these molecules near the repair interface contributes to functional integration.     

Effect of human interleukin-1 on cartilage metabolism

This study was undertaken to elucidate the effect of human interleukin-1 (IL-1) on the cartilage metabolism, with H235SO4 and 3H-proline as the respective indices for the metabolism of proteoglycan and collagen. We examined in vitro the 35S and 3H rates of incorporation after IL-1 was added to free chondrocytes incubated from human and porcine joint cartilage. As a result, IL-1 suppressed dose-dependently the secretion of both 35S and 3H into the supernatant of the medium containing free human or porcine chondrocytes. There was no difference between the control and the IL-1 group in the elution pattern of proteoglycan on the column-chromatogram. These results indicated that human IL-1 did not affect the molecular weight of the proteoglycan produced by human and porcine chondrocytes.     

Effects of polymethylmethacrylate on rabbit articular chondrocytes in monolayer culture

The effects of polymethylmethacrylate (PMMA) on DNA, protein, and sulfated-proteoglycan synthesis by rabbit articular chondrocytes were observed in monolayer cultures. PMMA pellets in ratios of 1:1 and 1:2 (liquid monomer:powder) significantly reduced [3H]thymidine incorporation into DNA during the first 24 h of culture and less so after 48 and 72 h. The reduction in [3H]thymidine incorporation was restricted to the cohort of chondrocytes nearest the PMMA. Consequently, cellular proliferation was unaltered by PMMA. By contrast, PMMA failed to inhibit [3H]leucine or [3H]serine/35SO4 incorporation. Both control and PMMA (1:1)-treated chondrocyte CsCl density gradient medium fraction dA1 eluted as a retarded peak on Sepharose CL-2B under associative conditions. The average partition coefficient (Kav) of PMMA-treated fraction dA1 was 0.41, as compared with 0.27 for control cultures. The Kav of medium fraction dD1 (proteoglycan monomer) was unaltered. Both control and PMMA-treated dA1 fraction elution profiles on Sepharose CL-2B were altered by incubation with Streptomyces hyaluronidase, indicating the presence of proteoglycan aggregate. The PMMA-treated cultures synthesized smaller proteoglycan aggregates. Since PMMA has been a critical factor in the success of total joint arthroplasty, defining interactions of differentiated cells with the cement is imperative for an understanding of the effects of PMMA on the biology of cartilage and bone.     

Effect of static compression on proteoglycan biosynthesis by chondrocytes transplanted to articular cartilage in vitro

Transplantation of chondrocytes by injection or within carrier matrices has shown promise for augmenting the repair of articular cartilage defects. In vivo, transplanted chondrocytes are exposed to mechanical forces. This in vitro study examined the effect of a step application of compressive load to chondrocytes after the cells had been seeded onto a cartilage surface. Bovine chondrocytes were transplanted onto bovine cartilage disks, allowed to attach for 1 hour or 4 days, and subjected to compression through overlying cartilage disks in a confined compression configuration. Before use, the disks were lyophilized to lyse the endogenous chondrocytes and thereby allow assessment of the metabolic activity of the transplanted cells. During a 16-hour application of compressive stress of 0-24-0.72 MPa, proteoglycan synthesis, assessed as [³⁵S]sulfate incorporation into macromolecules. was inhibited by approximately 68% after the 1-hour attachment and by approximately 45% after the 4-day attachment. Cell retention after the application of load was assessed by use of [³H]thymidine-tagged chondrocytes and quantitation of the displacement of radioactivity. After the 1-hour seeding period, loading induced a dose-dependent dislodgment of [³H]radioactivity (as much as 35%) from the tissue bilayer. In contrast, after the 4-day seeding period, there was no detectable effect of loading on chondrocyte dislodgment with an 8–12% release of radioactivity. The inhibitory effect of a 16-hour compression of 0.48 MPa applied after the 4-day seeding period was studied further. This protocol did not appear to have an irreversible effect on chondrocyte metabolism; at 2 days after the release of load, proteoglycan synthesis by the loaded cells was stimulated by 41% compared with transplanted cells that were not subjected to loading. These results suggest that the application of static compressive stress to chondrocytes at a cartilage surface may affect biosynthesis by these cells and thus subsequent integrative cartilage repair. Such an effect may have implications for optimization of the tightness of the press fit of a cell-laden cartilaginous construct into an articular defect.     

Influence of intraarticular continuous perfusion of physiological saline on chondrocytes

Physiological saline, which is generally used in arthroscopic examination and operation, is not physiological in the synovial joints. This paper discusses metabolic changes in the articular cartilage following continuous intraarticular perfusion with physiological saline, in comparison with in vitro findings. Fourty rabbits weighing approximately 3 kg were intraarticularly and continuously perfused with saline at a rate of 250 ml/hour. Articular cartilage of the femoral condyle was collected for measurement of 35SO4 radioactivity by liquid-scintillation spectrometry. Incorporation of 35SO4 showed increment in perfused articular cartilage as compared with the controls after one hour. Namely, glycosaminoglycan synthesis of chondrocytes was accelerated temporarily by continuous perfusion of saline, and it was recovered twenty-four hours after perfusion. In addition, histological and biomechanical test showed no remarkable changes. But, these effects will be expanded by the presence of degenerated articular cartilage. Continuous perfusion of physiological saline changes the physiological circumstance for articular cartilage, and influences on the chondrocytes' metabolism temporarily.     

In vitro growth of bovine articular cartilage chondrocytes in various capacitively coupled electrical fields

Isolated articular cartilage chondrocytes from 1- to 3-week-old male Holstein calf knee joints were formed into pellets containing 4 X 10(6) isolated cells and were grown in tissue culture medium (minimum essential medium/NCTC 135) containing either 1 or 10% newborn calf serum (NBCS) in plastic Petri dishes in 5% CO2 and air at 37 degrees C in saturation humidity. On the 4th postisolation day either [35S]sulfate or [3H]thymidine was added to the medium, and the pellets were exposed for 24 h to capacitively coupled electrical fields (10, 100, 250, and 1,000 V peak-to-peak, 60 kHz, sine wave signals). The pellets were then harvested, dialyzed, hydrolyzed, and assayed for DNA, protein, [35S]sulfate incorporation, and [3H]thymidine incorporation. Results indicated that at 250 V peak-to-peak there was a statistically significant increase in [35S]sulfate in 1% NBCS and a statistically significant increase in [3H]thymidine in 10% NBCS. At potentials above or below 250 V no changes were noted. Thus, articular cartilage chondrocytes grown in pellet form can be stimulated to increase glycosaminoglycan synthesis or to increase cell proliferation by an appropriate capacitively coupled electrical field. The importance of the serum concentration in the medium in evaluation of biosynthesis in vitro is noted.     

利用组织工程技术再生软骨组织的实验研究

目的 在有免疫力的动物体兔体内探索组织工程化软骨生成的影响因素。 方法 经不同物质修猸的聚羟基乙酸支架与软骨细胞体外培养,观察基质产生情况,并将细胞支架复合物体内回植,观察软骨的生成,并进行组织学及超微结构评价。结果 以孵磷脂、多聚赖氨酸及聚乳酸共同修饰的聚羟基乙酸与软骨细胞体外培养,结果基质产生旺盛,体内回植后软骨生成良好。 结论 细胞支架复合物体外培养期间有基质产生,是组织工程化软骨生成的前提     

骨髓基质细胞源性软骨细胞修复兔全层关节软骨缺损

目的观察体外诱导骨髓基质细胞(MSCs)源性软骨细胞在兔股骨滑车关节面全层软骨缺损修复中的作用. 方法高密度传代培养第3代诱导MSCs分化为软骨细胞,以酸溶性Ⅰ型胶原为载体,两者混合后形成凝胶样植入物(细胞浓度为5×106/ml).于36只新西兰大耳白兔一侧股骨滑车关节面造成3 mm×5 mm全层关节软骨缺损,凝胶样植入为实验侧;另一侧分别为单纯胶原植入组(18个膝关节)和空白对照组(18个膝关节).术后4、8、12、24、32和48周取材观察缺损修复情况及新生组织的类型.参照Pineda标准对新生组织评分. 结果实验侧术后4周,植入细胞类似软骨细胞,周围有异染基质,形成透明软骨样组织;8周,深层有软骨下骨形成,软骨细胞层较正常关节软骨厚;12周,新生软骨厚度减小,与正常软骨相近,细胞呈柱状排列,结构与正常关节软骨相似,软骨下骨形成,潮线恢复;24周,新生软骨厚度较正常薄,约占55%,表面平整,潮线附近仍有肥大的软骨细胞;32周,潮线附近无肥大软骨细胞;48周,组织结构与32周时基本相同,为类透明软骨.Pineda评分24、32和48周间无差异,与4周比较有统计学意义(P＜0.05).实验组2～48周期间关节功能良好.单纯胶原组与空白对照组缺损无修复,48周时软骨下骨外露,关节退变;关节功能逐渐减退,动度受限. 结论 MSCs源性软骨细胞移植体内可形成透明样软骨组织,24周后新生软骨特性稳定,48周时为透明样软骨,能维持良好的关节功能.     

Metabolic and structural changes in newly synthesized proteoglycans induced by implantation of a polyethylene sheet in the rabbit knee joint

A sheet of polyethylene was surgically implanted in a rabbit right patello-femoral joint and changes in the structure and chemical composition of newly synthesized articular cartilage proteoglycans (PGs) were studied 1 month after surgery. The articular cartilage from implanted and sham-operated control knee joints was labeled in vitro with 3H-glycine and 35S-SO4 and then extracted with 4 M guanidinium chloride (GuHCl) solution. Labeled extracts were analyzed by dissociative CsCl gradient centrifugation and by Sepharose CL-2B column chromatography. The labeled glycosaminoglycan side chains were analyzed by Sephadex G-200 column chromatography and specific enzymatic digestions. Compared with sham-operation, the trochlear articular cartilage of operated joints incorporated more 35S-SO4 and 3H-glycine into newly synthesized PGs and proteins. It also synthesized a higher proportion of extractable, hydrodynamically large and high density 35S-PG monomers with increased proportion of molecules, able to interact with exogeneous hyaluronan (HA). The fibro-cartilagenous 'osteo-chondrophytic' spurs, compared with trochlear hyaline articular cartilages, incorporated less 35S-SO4 and 3H-glycine and synthesized less extractable high density 35S-PG monomers able to interact with exogenous HA. Their 35S-GAG side chains were more heterogeneous and segregated into three distinct peaks as shown by Sephadex G-200 column chromatography. The results of the present studies demonstrate that, in response to the implant, there was an increase in the biosynthetic capacity of chondrocytes which synthetized larger PG monomers able to interact wih HA.     

BMP—2对培养兔关节软骨细胞代谢的影响

目的：探讨BMP－2对体外培养兔关节软骨细胞代谢影响进而了解其在关节软骨损伤修复中的作用。方法：利用荧光分光光度计,721－型分光光度计,^S-Na2SO4同位素掺入法等测定DNA含量、胶原和蛋白多糖的合成。结果：BMP－2既能促进原代及反分化关节软骨细胞DNA合成又能增加羟脯氨酸的含量^35S-Na2SO4掺入。结论：ＢＭＰ－２能促进软骨细胞的增殖和维持其表型,还可使已丢失软骨表型的反分化软骨细胞有向恢复软骨表型方向分化的可能。     

Evaluation of articular cartilage with 3D-SPGR MRI after autologous chondrocyte implantation

To assess the maturation process of the cartilage after autologous chondrocyte implantation (ACI), we performed a longitudinal study with three-dimensional spoiled gradient-recalled (3D-SPGR) magnetic resonance imaging (MRI). Five knees of five patients on which ACI of the femoral condyle was performed were studied. The signal intensity of reparative tissue approached that of normal articular cartilage with time. The volume of reparative tissue remained at an almost constant level after implantation. During second-look arthroscopy, the areas on which ACI was performed were covered with hyaline-like cartilage, and the reparative tissue removed by biopsy consisted of normal chondrocytes and extracellular matrix. The increased signal intensity of the reparative tissue represents maturation of implanted autologous chondrocytes. 3D-SPGR MRI is thought to be useful for evaluating reparative tissue after autologous chondrocyte implantation.     

Low frequency EMF regulates chondrocyte differentiation and expression of matrix proteins.

This study describes the enhancement of chondrogenic differentiation in endochondral ossification by extremely low frequency pulsed electric/magnetic fields (EMFs). The demineralized bone matrix (DBM)-induced endochondral ossification model was used to examine the effects of EMF stimulation. [35S]-Sulfate and [3H]-thymidine incorporation and glycosaminoglycan (GAG) content were determined by standard methods. Proteoglycan (PG) and GAG molecular size and composition were determined by gel chromatography and sequential enzyme digestion. Immunohistochemical and Western blot analysis of PGs were done with antibodies 2B6, 3B3, 2D3 and 5D4. Northern analysis of total RNA extracts was performed for aggrecan, and type II collagen. All data was compared for significance by Student's t- or analysis of variance (ANOVA)-tests. The EMF field accelerated chondrogenesis as evidenced by an increase in: (1) 35SO4 incorporation and GAG content, (2) the number of chondrocytes at day 8 of development, (3) the volumetric density of cartilage and (4) the extent of immunostaining for 3B3 and 5D4. No differences in DNA content or [3H]-thymidine incorporation were observed between control and stimulated ossicles, suggesting the absence of enhanced cell proliferation or recruitment as a mechanism for the acceleration. PG and GAG molecular sizes and GAG chemical composition were similar in stimulated and control ossicles, indicating that stimulation resulted in an accelerated synthesis of normal cartilage molecules. The increased expression of PG and type II collagen mRNA as well as a greater immunoreactivity of 3B3 and 5D4 suggest an increase in the rate of differentiation of chondrocytes and enhanced phenotypic maturation.     

Demonstration of increased proteoglycan turnover in cartilage explants from dogs with experimental osteoarthritis

The turnover of proteoglycans (assessed by the release into the medium of newly synthesised [35S]-proteoglycan) in explant cultures of articular cartilage from various anatomical sites of the knee joints (stifle) of mature beagles with experimental osteoarthritis has been studied with the following findings: (a) The proportion of newly synthesised proteoglycans released from cartilage explants maintained in vitro was generally increased for cartilage from operated compared with nonoperated control joints. (b) At 3 weeks after surgery there was a significant increase in the release of [35S]-proteoglycans from explants of the lateral and medial tibial plateaux of operated joints compared with sham-operated joints but not from other sites. On the other hand, when this comparison was made at 3 to 6 months after surgery, significant increases in the release of [35S]-proteoglycans were observed from cartilage of all anatomical areas except the patellar groove. (c) The release of [35S]-proteoglycan from cartilage explant cultures was dependent on live chondrocytes, since freeze-thawing the tissue immediately after labelling markedly reduced the release from both normal and osteoarthritic cartilage.     

Prolonged-fresh preservation of intact whole canine femoral condyles for the potential use as osteochondral allografts.

Defects in articular cartilage are often repaired with fresh osteochondral grafts. While fresh allografts provide viable chondrocytes, logistic limitations require surgical implantation within seven days of graft harvest. Here, we provide information on cold preservation of whole intact osteochondral materials that retains cartilage cell viability and function, and histologic and biochemical integrity for 28 days. Canine femoral condyles were obtained and stored at 4 degrees C for 14, 21 or 28 days. At the end of the storage period, cartilage was assessed for cell viability, 35S uptake, proteoglycan content and histologic parameters. The most noticeable histologic change was reduced Safranin-O near the cartilage surface with 14 days of cold preservation, but had recovered with 21 and 28 days. Cartilage thicknesses did not vary significantly. Cell viability was >95% at 14 days, 75-98% at 21 days and reduced to 65-90% at 28 days. Cell function measures showed that the level of 35SO4 incorporation was suppressed in samples stored at 4 degrees C. However, no significant differences were seen among groups at 14, 21 or 28 days of cold preservation. This data has implications for tissue banking protocols for osteochondral allograft material obtained for transplantation suggesting that cold preserved allograft material be implanted within 28 days.     

Variation of cell and matrix morphologies in articular cartilage among locations in the adult human knee

OBJECTIVE: Understanding of articular cartilage physiology, remodelling mechanisms, and evaluation of tissue engineering repair methods requires reference information regarding normal structural organization. Our goals were to examine the variation of cartilage cell and matrix morphology in different topographical areas of the adult human knee joint. METHODS: Osteochondral explants were acquired from seven distinct anatomical locations of the knee joints of deceased persons aged 20-40 years and prepared for analysis of cell, matrix and tissue morphology using confocal microscopy and unbiased stereological methods. Differences between locations were identified by statistical analysis. RESULTS: Medial femoral condyle cartilage had relatively high cell surface area per unit tissue volume in the superficial zone. In the transitional zone, meniscus-covered lateral tibia cartilage showed elevated chondrocyte densities compared to the rest of the knee while lateral femoral condyle cartilage exhibited particularly large chondrocytes. Statistical analyses indicated highly uniform morphology throughout the radial zone (lower 80% of cartilage thickness) in the knee, and strong similarities in cell and matrix morphologies among cartilage from the femoral condyles and also in the mediocentral tibial plateau. Throughout the adult human knee, the mean matrix volume per chondron was remarkably constant at approximately 224,000 microm(3), corresponding to approximately 4.6 x 10(6) chondrons per cm(3). CONCLUSIONS: The uniformity of matrix volume per chondron throughout the adult human knee suggests that cell-scale biophysical and metabolic constraints may place limitations on cartilage thickness, mechanical properties, and remodelling mechanisms. Data may also aid the evaluation of cartilage tissue engineering treatments in a site-specific manner. Results indicate that joint locations which perform similar biomechanical functions have similar cell and matrix morphologies; findings may therefore also provide clues to understanding conditions under which focal lesions leading to osteoarthritis may occur.     

Degree of differentiation of chondrocytes and their pretreatment with platelet-derived-growth factor. Regulating induction of cartilage formation in resorbable tissue carriers in vivo

Current methods for articular cartilage repair are unpredictable with respect to clinical success. In the present study, we investigated the ability of cells from articular cartilage, perichondrium, and costochondral resting zone to form new cartilage when loaded onto biodegradable scaffolds and implanted into calf muscle pouches of nu/nu mice. Prior in vitro studies showed that platelet derived growth factor-BB (PDGF-BB), but not transforming growth factor beta-1 (TGF-beta 1), basic fibroblast growth factor, or bone morphogenetic protein-2 promoted proliferation and extracellular matrix sulfation of resting zone chondrocytes without causing the cells to exhibit a hypertrophic chondrocyte phenotype. TGF-beta 1 has also been shown to stimulate chondrogenesis by multipotent chondroprogenitor cells like those in the perichondrium. In addition, PDGF-BB has been shown to modulate chondrogensis by resting zone cells implanted in poly(D,L-lactide-co-glycolide) (PLG) scaffolds. In the present study we examined whether the cartilage formation is dependent on state of chondrocyte maturation and whether the pretreatment of chondrocytes with growth factors has an influence on the cartilage formation. Scaffolds were manufactured from 80% PLG with a 75:25 lactide:glycolide ratio and 20% modified PLG with a 50:50 lactide:glycolide ratio (PLG-H scaffolds). For each experimental group, four nude mice received two identical implants, one in each calf muscle resulting in an N = 8 implants: PLG-H scaffolds alone; PLG-H scaffolds with cells derived from either the femoral articular cartilage, costochondral periochondrium, or costochondral resting zone cartilage of 125 g male Sprague-Dawley rats; PLG-H scaffolds with either articular chondrocytes or resting zone chondrocytes that were pretreated with 37.5 ng/ml rhPDGF-BB for 4 h or 24 h before implantation, or with perichondrial cells treated with PDGF-BB plus 0.22 ng/ml rhTGF beta-1 for 4 h and 24 h. At 4 or 8 weeks after implantation, samples were harvested and analyzed histomorphometrically for new cartilage formed, area of residual implant and area of fibrous connective tissue. Only resting zone cells showed the ability to form new cartilage at a heterotopic site in this study. There was no neocartilage found in nude mice with implants loaded with either articular chondrocytes or perichondrial cells. Pretreatment of resting zone chondrocytes for 4 h prior to implantation significantly increased the amount of newly formed cartilage after 8 weeks and suppressed chondrocyte hypertrophy. The amount of fibrous connective tissue around implants containing either articular chondrocytes or perichondrial cells decreased with time, whereas the amount of fibrous connective tissue around implants containing resting zone chondrocytes pretreated with PDGF-BB was increased. The results showed that resting zone cells can be successfully incorporated into biodegradable porous PLG scaffolds and can induce new cartilage formation in a nonweight-bearing site. Articular chondrocytes as well as perichondrial cells did not have the capacity for neochondrogenesis when implanted heterotopically in this model.     

Cartilage-derived morphogenetic protein-1 and -2 are endogenously expressed in healthy and osteoarthritic human articular chondrocytes and stimulate matrix synthesis

OBJECTIVE: We investigated whether chondrocytes derived from osteoarthritic cartilage may lose their responsiveness to cartilage-derived morphogenetic protein-1, -2 (CDMP-1, -2) and osteogenic protein-1 (OP-1) compared with healthy cells, thus leading to an impaired maintenance of matrix integrity. DESIGN: Chondrocytes were isolated from articular cartilage from patients with and without osteoarthritic lesions. Cells were grown as monolayer cultures for 7 days in a chemically defined serum-free basal medium (BM) in the presence of recombinant CDMP-1, -2, and OP-1. Glycosaminoglycan synthesis was measured by [35S]Sulfate incorporation into newly synthesized macromolecules. Cell proliferation was investigated by [3H]Thymidine incorporation. The endogenous gene expression of CDMPs/OP-1 and their respective type I and type II receptors was examined using RT-PCR. The presence of CDMP proteins in tissue and cultured cells was detected by Western immunoblots. RESULTS: mRNAs coding for CDMPs and their respective receptors are endogenously expressed not only in healthy, but also in osteoarthritic cartilage. CDMP proteins are present in both normal and osteoarthritic articular cartilage and cultured chondrocytes. CDMP-1, CDMP-2 and OP-1 markedly increased glycosaminoglycan synthesis in both healthy (P< 0.01) and osteoarthritic (P< 0.05) human articular chondrocytes. A comparison of the glycosaminoglycan biosynthetic activity between healthy and osteoarthritic samples revealed no detectable difference, neither in stimulated nor in unstimulated cultures. [(3)H]Thymidine incorporation showed that CDMPs/OP-1 did not affect cell proliferation in vitro. CONCLUSION: CDMPs and OP-1 exert their anabolic effects on both healthy and osteoarthritic chondrocytes indicating no loss in responsiveness to these growth factors in OA. The endogenous expression of CDMPs/OP-1 and their receptors suggest an important role in cartilage homeostasis.     

Growth-plate cartilage metabolic response to mechanical stress

An in vitro study assessing the metabolic response of growth-plate cartilage explants to mechanical stress was performed. Cultured explants were exposed to two types of stress: (a) single high-compressive force (SHC), and (b) multiple intermittent low-compressive force (MILC). Proliferative activity and matrix synthesis were determined with liquid scintillation counting after explants were labeled with [3H]thymidine and [35S]-sulfate. Our in vitro findings suggest that too high a force, even acting for a short time, may result in permanent injury of growth plate cartilage. Hence, we hypothesized that there might be an unknown pathogenetic mechanism of Salter's fifth-type epiphyseal injury. We report the results of our study.     

Study of the localization of [3H]Bovine parathyroid hormone in bone by light microscope autoradiography

Summary Bovine parathyroid hormone labeled with tritium on the methionine residues by [³H]methyl exchange ([³H]bPTH) was administered intravenously to 35-day-old mice and localized in tissues by light microscope autoradiography. After 10 min most of the [³H]bPTH had been taken up from plasma. In the kidney and liver, radioactivity was not displaced by simltaneous administration of unlabeled bPTH, and was as intense after giving oxidized [³H]bPTH ([³H]ox bPTH) as [³H]bPTH, suggesting that binding was largely associated with nonspecific processes. In long bones, [³H]bPTH was bound to osteoblasts and preosteoblasts lining the endosteal and periosteal surfaces of compact bone. In the area of the growth-plate there was intense labeling on new endochondrial bone, and on hypertrophic chondrocytes in the region of calcification. There was little labeling in marrow and, in contrast to other tissues, much reduced binding was seen when a large excess of unlabeled parathyroid hormone was administered together with [³H]-bPTH, or when [³H]ox bPTH was given. It is concluded that binding of parathyroid hormone to cells in bone is largely specific, and that the hormone has a function in cartilage which relates to the differentiation and calcification of chondrocytes of the growth-plate that occurs prior to its replacement by new bone tissue.     

应用细胞膜片复合硅胶管内支撑构建组织工程管状软骨

目的 研究利用羊耳软骨细胞形成细胞膜片,构建无细胞支架组织工程管状软骨的可行性.方法 体外培养扩增羊耳软骨细胞至第2代,以2.0×106 cells/mL的高密度,培养7d,形成直径为35 mm的圆形细胞膜片,将细胞膜片均匀包裹于硅胶管,植入裸鼠背部皮下,4周后取材检测.以大体观察、组织学、免疫组织化学等方法,对形成的软骨进行评价.结果 大体观察可见形成良好的管状软骨,HE染色见软骨陷窝形态规则,番红O染色及Ⅱ型胶原免疫组化均呈阳性表达.结论 细胞膜片复合硅胶管内支撑的方法能形成管状软骨,为气管软骨的再造提供了新的方法.