Osteogenic protein-1 promotes the formation of tissue-engineered cartilage using the alginate-recovered-chondrocyte method

OBJECTIVE: This study examined the effects of a growth factor, recombinant human osteogenic protein-1 (rhOP-1), on the formation of tissue-engineered cartilaginous tissue by adult bovine articular chondrocytes using the alginate-recovered-chondrocyte (ARC) method. DESIGN: To ascertain if rhOP-1 enhances the formation of the cell-associated matrix (CM) and the characteristics of CM formation, bovine articular chondrocytes were first cultured for up to 14 days in alginate beads in medium supplemented with serum, with or without rhOP-1. Then, the recovered chondrocytes and their associated CM were resuspended in medium, with or without OP-1, seeded onto culture inserts, and incubated for an additional 14 days. The fabricated ARC tissues were subjected to biochemical and histological analyses. RESULTS: The addition of rhOP-1 to the medium in the alginate bead culture step resulted in an increased accumulation of both proteoglycan (PG) and collagen, with a ratio of PG to collagen that was higher than that found in native adult cartilage. The addition of rhOP-1 in the second step had a similar stimulatory effect during 14 days of culture. Histological examination of the tissue formed under all conditions revealed a cartilage-like matrix, stained strongly by toluidine blue. The thickness of the tissues obtained from culture conditions that included the addition of rhOP-1 was four times greater than that of the tissues cultured without rhOP-1. CONCLUSIONS: Using the ARC method, rhOP-1 enhanced the formation of matrix and generated a voluminous tissue-engineered cartilaginous construct. These characteristics may be beneficial in generating constructs that can cover large defects.     

Novel ultrasonic evaluation of tissue-engineered cartilage for large osteochondral defects--non-invasive judgment of tissue-engineered cartilage.

Although numerous methods for regenerating articular cartilage have been investigated, the regenerated tissue showed various histological findings from hyaline-like cartilage to fibrous tissue. Without biopsy, we are unable to know whether the cartilage regeneration method was histologically successful or not. We developed a new ultrasonic evaluation system for articular cartilage using the maximum magnitude (MM) from ultrasonic analysis. The purpose of this study was to investigate the usefulness of ultrasonic judgment of the cartilage regeneration procedure. Using our system we quantitatively evaluated tissue-engineered cartilage in rabbit cartilage defects. The specimens were retrospectively divided into two groups on the basis of histological findings and investigated whether significant differences in ultrasonic analysis could be found between the two (group H: hyaline-like cartilage group, successful; group F: fibrous tissue group, failure). In the ultrasonic findings, the MM was 1.11+/-0.32 in group H and 0.65+/-0.18 in group F and these differences were significant (P=0.00061). Our results suggest that the ultrasonic evaluation system used in the present study is capable of judging the success or failure of cartilage regeneration procedures, and therefore, it could be a valuable tool arthroscopic diagnosis of cartilage regeneration.     

三种支架材料与软骨细胞复合构建组织工程化软骨的研究

目的：研究细胞、材料及孔径在诱导关节软骨发育过程中的作用。方法：采用细胞与支架的三维培养和体内植入技术。结果：种入支架的原代软骨细胞增殖差，未形成软骨组织。而第2代软骨细胞生长良好，最终发育成软骨组织。孔径为25－100μm的胶原海绵捕捉的细胞量明显多于孔径为100－550μm的明胶海绵和PDLLA泡沫。裸鼠皮下植入16周时3种支架－细胞复合体比较，胶原海绵－细胞复合体中的支架材料降解最彻底，软骨组织发育最好。结论：第3代软骨细胞细胞增殖优于原代细胞；25－100μm的小孔径支架对细胞的吸附量优于100－550μm的大孔径支架；软骨发育中支架材料的彻底降解是优质软骨组织形成的前提。     

Characterization of chondrocytes from bovine articular cartilage: I. Metabolic and morphological experimental studies

Articular chondrocytes were enzymatically isolated from mature bovine cartilage and separated by density zone-velocity sedimentation in a gradient. For cells from different levels of the gradient, size and staining characteristics were determined and rates of incorporation of tritiated cytidine and radiosulphate were measured as indicators of RNA and sulphated glycosaminoglycan synthesis, respectively. The data clearly show that the chondrocyte population is composed of cells that vary continuously in size and metabolic activity from one limit to another. The largest cells also demonstrated the greatest RNA production while the smallest cells had the least. There was, however, no such differentiation of sulphated proteoglycan production.     

A novel method for determination of collagen orientation in cartilage by Fourier transform infrared imaging spectroscopy (FT-IRIS)

OBJECTIVE: The orientation of collagen molecules is an important determinant of their functionality in connective tissues. The objective of the current study is to establish a method to determine the alignment of collagen molecules in histological sections of cartilage by polarized Fourier transform infrared imaging spectroscopy (FT-IRIS), a method based on molecular vibrations. METHODS: Polarized FT-IRIS data obtained from highly oriented tendon collagen were utilized to calibrate the derived spectral parameters. The ratio of the integrated areas of the collagen amide I/II absorbances was used as an indicator of collagen orientation. These data were then applied to FT-IRIS analysis of the orientation of collagen molecules in equine articular cartilage, in equine repair cartilage after microfracture treatment, and in human osteoarthritic cartilage. Polarized light microscopy (PLM), the most frequently utilized technique to evaluate collagen fibril orientation in histological sections, was performed on picrosirius red-stained sections for comparison. RESULTS AND CONCLUSION: Thicknesses of each zone of normal equine cartilage (calculated based on differences in collagen orientation) were equivalent as determined by PLM and FT-IRIS. Comparable outcomes were obtained from the PLM and FT-IRIS analyses of repair and osteoarthritis tissues, whereby similar zonal variations in collagen orientation were apparent for the two methods. However, the PLM images of human osteoarthritic cartilage showed less obvious zonal discrimination and orientation compared to the FT-IRIS images, possibly attributable to the FT-IRIS method detecting molecular orientation changes prior to their manifestation at the microscopic level.     

Zone-specific cell biosynthetic activity in mature bovine articular cartilage: A new method using confocal microscopic stereology and quantitative autoradiography

A new methodology was developed to measure spatial variations in chondrocyte/matrix structural parameters and chondrocyte biosynthetic activity in articular cartilage. This technique is based on the use of a laser scanning confocal microscope that can “optically” section chemically fixed, unembedded tissue. The confocal images are used for morphometric measurement of stereologic parameters such as cell density (cells/mm³), cell volume fraction (%), surface density (1/cm), mean cell volume (μm³), and mean cell surface area (μm²). Adjacent pieces of tissue are simultaneously processed for conventional liquid emulsion autoradiography, and a semiautomated grain counting program is used to measure the silver grain density at regions corresponding to the same sites used for structural measurements. An estimate of chondrocyte biosynthetic activity in terms of grains per cell is obtained by dividing the value for grain density by that for cell density. In this paper, the newly developed methodology was applied to characterize the zone-specific behavior of adult articular cartilage in the free-swelling state. Cylinders of young adult bovine articular cartilage were labelled with either [³H]proline or [³⁵S]sulfate, and chondrocyte biosynthesis and structural parameters were measured from the articular surface to the tidemark. The results showed that chondrocytes of the radial zone occupied twice the volume and surface area of the chondrocytes of the superficial zone but were 10 times more synthetically active. This efficient and unbiased technique may prove useful in studying the correlation between mechanically induced changes in cell form and biosynthetic activity within inhomogeneous tissue as well as metabolic changes in cartilage due to ageing and disease.     

软骨细胞诱导骨髓基质干细胞构建带内支撑的组织工程化软骨

目的 探讨以聚羟基乙酸(PGA)包裹特定形态的医用假体材料--多孔高密度聚乙烯(HDPE,商品名为MEDPOR)为支架,应用软骨细胞诱导骨髓基质干细胞(BMSCs),共培养构建特定形态的带内支撑组织工程化软骨医用假体的可能性.方法 以直径3 mm、长5 mm的圆柱形HDPE,外裹 1 mm厚PGA为支架,将体外分别培养的新生猪BMSCs和耳郭软骨细胞按7∶3混合,以10×10 7/ml细胞浓度接种于支架上,同时以相同浓度的单纯软骨细胞和单纯BMSCs分别接种,作为阳性对照组(PC组)和阴性对照组(NC组).经体外培养2周及在裸鼠皮下移植4、8周后取材 ,行大体观察、组织学、组织化学及免疫组化检测.结果 各组细胞均与材料黏附良好.实验组和阳性对照组均形成了大体形态良好的HDPE-软骨复合体,内支撑的HDPE与外层软骨结合紧密.组织学可见成熟的软骨陷窝结构,软骨渗入HDPE孔隙内部、异染基质及Ⅱ型胶原呈强阳性表达.结论 以HDPE为内支撑,外裹PGA的支架,接种混合细胞,可于皮下构建特定形态、组织学良好的HDPE-软骨复合体.     

软骨细胞诱导骨髓基质干细胞构建带内支撑的组织工程化软骨

目的探讨以聚羟基乙酸(PGA)包裹特定形态的医用假体材料多孔高密度聚乙烯(HDPE,商品名为MEDPOR)为支架,应用软骨细胞诱导骨髓基质干细胞(BMSCs),共培养构建特定形态的带内支撑组织工程化软骨医用假体的可能性。方法以直径3mm、长5mm的圆柱形HDPE,外裹1mm厚PGA为支架,将体外分别培养的新生猪BMSCs和耳郭软骨细胞按7:3混合,以10×107/ml细胞浓度接种于支架上,同时以相同浓度的单纯软骨细胞和单纯BMSCs分别接种,作为阳性对照组(PC组)和阴性对照组(NC组)。经体外培养2周及在裸鼠皮下移植4、8周后取材,行大体观察、组织学、组织化学及免疫组化检测。结果各组细胞均与材料黏附良好。实验组和阳性对照组均形成了大体形态良好的HDPE-软骨复合体,内支撑的HDPE与外层软骨结合紧密。组织学可见成熟的软骨陷窝结构,软骨渗入HDPE孔隙内部、异染基质及Ⅱ型胶原呈强阳性表达。结论以HDPE为内支撑,外裹PGA的支架,接种混合细胞,可于皮下构建特定形态、组织学良好的HDPE-软骨复合体。     

Localization of insulin-like growth factor binding protein-2 in chondrocytes of bovine articular cartilage.

PURPOSE: Previous work indicated that transforming growth factor (TGF-beta) treatment of bovine articular cartilage resulted in an accumulation of insulin-like growth factor binding protein-2 (IGF-BP-2). The purpose of the work presented in this paper was to define the localization of the IGF-BP-2 in freshly excised articular cartilage and in slices cultured in the presence and absence of TGF-beta. METHOD: Newborn calf articular cartilage was dissected and immediately fixed or maintained in organ culture for five days under basal conditions (media without added serum or growth factors) or with basal media containing 15 ng/ml of TGF-beta1. Frozen or paraffin embedded sections were prepared, and immunohistochemistry using anti-IGF-BP-2 performed. RESULTS: The paraffin sections provided the best preservation of morphology and consistency of immunohistochemical staining patterns. In fresh cartilage slices, IGF-BP-2 was associated with most of the chondrocytes. The basal cultured cartilage showed positive immunostaining in some areas, but not others: the most consistently stained area was the upper radial zone. In all cases where a positive reaction was observed, it was associated mostly with chondrocytes. On the other hand, all the TGF-beta treated samples that were examined in this study were evenly stained, and most chondrocytes were positive in all areas from superficial to deep zones, thus closely resembling the pattern of fresh tissue. CONCLUSIONS: It is concluded that IGF-BP-2 is closely cell associated in bovine articular cartilage. Following culture of cartilage slices, TGF-beta increases the number of cells with positive immunostaining. These data help to support the postulate that TGF-beta exerts at least some of its actions in articular cartilage via cross-talk mechanisms involving the IGF-BP-2 system.     

Normal features of tissue-engineered auricular cartilage by flow cytometry and histology: patient safety.

BACKGROUND: Cytokinetic abnormalities in DNA content, such as aneuploidy, haploidy, and tetraploidy, have been found to occur in human cartilaginous tumors. The high number of chondrocytes needed for tissue-engineered cartilaginous implants requires the cells to be passaged repeatedly. The theoretical risk of changes in the normal diploid state of these cells during their growth in vitro and after generation of tissue-engineered cartilage in vivo is not known.Materials and methods Auricular chondrocytes were obtained from 6 patients and cultured in vitro. Chondrocyte number was increased by repeated passaging. The passaged cells were implanted in nude mice for 8 weeks to generate tissue-engineered cartilage. Fresh control chondrocytes along with the passaged cells and cells obtained from the tissue-engineered constructs were collected and compared for DNA content by flow cytometry. RESULTS: Flow cytometry demonstrated 100% diploidy with no evidence of aneuploidy, haploidy, or tetraploidy in all groups of cells. Histology of the tissue-engineered cartilage also showed no evidence of cellular atypia. CONCLUSION: The number of human auricular chondrocytes can be increased by repeated passaging and passaged chondrocytes can be safely used for implantation to generate tissue-engineered constructs without a change in the normal diploid state of the cells. Histology of the cartilage generated showed normal features without atypia.     

仿生组织工程软骨修复关节软骨损伤的效果及应用超声弹性成像方法检测价值分析

目的体外构建的仿生组织工程软骨修复羊膝关节软骨损伤,通过组织学染色、点压力学分析、超声弹性成像检查来评价修复效果,探讨超声弹性成像方法评估再生软骨质量的可行性。 方法共选用雄性山羊12只,随机分A、B、C三组,A组空白对照组2只,仅做股骨髁负重区直径6 mm全层软骨缺损;B组单纯仿生软骨（ECM）支架修复组4只,在股骨髁负重区软骨缺损处仅植入仿生软骨支架;C组仿生软骨支架复合自体骨髓间充质干细胞（BMSCs）组6只。取自体髂骨骨髓血分离骨髓间充质干细胞培养,细胞浓度达到1×10⁷个/毫升后加入仿生软骨支架中,将构建好的仿生组织工程软骨植入股骨髁软骨缺损中。每组的羊均先在左膝造模,术后3个月再右膝造模,待右膝术后3个月时先行超声弹性成像检查,然后取材行评分,病理染色、糖胺多糖（GAG）含量测定、力学检测分析。 结果3个月和6个月的A组基本无软骨组织修复。在大体评分、病理评分、GAG含量测定、点压力学测试的结果显示C组优于B组（P<0.05）,并且B组与C组中的6月数据均明显优于3个月（P<0.05）。其中C组动物6个月取材发现重建软骨更接近正常软骨组织。修复组织大体形态结果,点压力学测试结果与超声弹性成像检查结果对比基本一致。 结论利用组织工程方法构建仿生软骨可治疗早期软骨损伤,复合自体骨髓间充质干细胞的仿生支架能达到更好的修复效果。软骨损伤经过仿生软骨修复后恢复周期在6个月以上。超声弹性成像检查软骨修复效果可作为术后复查简单快捷有效的方法。     

A novel method for faster formation of rat liver cell spheroids

Hepatocyte spheroids are expected to be the main component of the artificial liver bioreactor for their higher function. The preparation of hepatocyte spheroids, however, can require as many as 24 to 96 h. To reduce this time, we investigated a method employing a new technique of rat hepatocyte preparation and a dynamic culture. The modified Seglen's method for standard hepatocyte isolation was altered by elimination of ethyleneglycol bis(aminoethylether) tetraacetate from the first perfusate and calcium from the second perfusate. Isolated hepatocytes were cultured in a spinner flask by spinning at 120 rpm. The modified Seglen's method was used as a control. Cells obtained by the new method were more cohesive and formed a higher proportion of cell aggregates than control cells. In the spinning culture, hepatocytes had a tendency to aggregate and 80% of cells formed spheroids within 6 h of culturing. The mean size of spheroids was 68.5 +/- 18.5 microm. Confocal laser scanning microscopy revealed that individual spheroids contained approximately 30% of nonparenchymal cells over their surface. Using the new hepatocyte preparation method followed by a spinning culture, we were able to produce hepatocyte spheroids in as few as 6 h.     

The use of specific chondrocyte populations to modulate the properties of tissue-engineered cartilage.

Tissue engineering of articular cartilage is a promising alternative to the conventional approaches for cartilage repair. However, recent attempts to develop articular cartilage in vitro have proven to be difficult. The tissue formed in vitro may not accumulate enough extracellular matrix, and the resulting mechanical properties are only a fraction of the native tissue. We investigated whether using specific populations of chondrocytes would improve the properties of the cartilaginous tissue that was generated in vitro. Full-thickness (FT), mid-and-deep zone (MD), and deep-zone (DEEP) chondrocytes were isolated, placed on the surface of porous ceramic substrates and maintained in culture for eight weeks. Tissue developed from DEEP chondrocytes was thicker (FT: 0.94+/-0.03, MD: 0.88+/-0.04, DEEP: 2.4+/-0.1 mm) and had accumulated larger amounts of extracellular matrix (FT: 1.61+/-0.05, MD: 1.5+/-0.1, DEEP: 3.8+/-0.2 mg dry weight) than the tissues formed by the FT and MD chondrocytes. The tissue formed by the FT chondrocytes accumulated the greatest amount of collagen (FT: 211+/-14, MD: 185+/-8, DEEP: 178+/-5 microg/mg dry weight) whereas the tissue formed by the MD chondrocytes accumulated significantly more proteoglycans (FT: 198+/-10, MD: 265+/-10, DEEP: 215+/-5 microg/mg dry weight). Interestingly, MD chondrocytes produced tissue that had compressive mechanical properties up to four times greater than the cartilaginous tissues formed by cells from either the FT or DEEP of cartilage. Thus, a combined population of intermediate and DEEP chondrocytes might be more suitable for the tissue engineering of articular cartilage.     

A histochemical method for the demonstration of calcifying cartilage

Summary The demonstration of histochemical characteristics in calcifying cartilage is fraught with methodological difficulties including the distinction of mineralized from unmineralized cartilage and the demonstration of cell detail in relatively hard tissue. This study uses the decalcified bone matrix-induced enchondral (endochondral) ossification system to demonstrate a technique of methylmethacrylate embedding, thin sections, and a combination of histochemical stains that distinguishes mineralized from unmineralized cartilage while preserving excellent cell detail. These techniques are applicable to other areas of enchondral ossification and are exemplified by the staining of growth plates.     

A method for morphometry of bone formation using fluorochromes (author's transl)

A reliable morphometric determination of newly formed bone requires a continuous labeling of the tissue of interest. A technique is described which uses the fluorochromes Calcein and Xylenolo-range as bone labels. Image contrast in the fluorescence microscope can be increased by means of special filter combinations to permit data collection by computer-compatible video-systems. This technique offers a wide field of applications in experimental bone biology.