Injectable tissue-engineered cartilage using a fibrin glue polymer.

The purpose of this study was to demonstrate the feasibility of using a fibrin glue polymer to produce injectable tissue-engineered cartilage and to determine the optimal fibrinogen and chondrocyte concentrations required to produce solid, homogeneous cartilage. The most favorable fibrinogen concentration was determined by measuring the rate of degradation of fibrin glue using varying concentrations of purified porcine fibrinogen. The fibrinogen was mixed with thrombin (50 U/cc in 40 mM calcium chloride) to produce fibrin glue. Swine chondrocytes were then suspended in the fibrinogen before the addition of thrombin. The chondrocyte/polymer constructs were injected into the subcutaneous tissue of nude mice using chondrocyte concentrations of 10, 25, and 40 million chondrocytes/cc of polymer (0.4-cc injections). At 6 and 12 weeks, the neocartilage was harvested and analyzed by histology, mass, glycosaminoglycan content, DNA content, and collagen type II content. Control groups consisted of nude mice injected with fibrin glue alone (without chondrocytes) and a separate group injected with chondrocytes suspended in saline only (40 million cells/cc in saline; 0.4-cc injections). The fibrinogen concentration with the most favorable rate of degradation was 80 mg/cc. Histologic analysis of the neocartilage showed solid, homogeneous cartilage when using 40 million chondrocytes/cc, both at 6 and 12 weeks. The 10 and 25 million chondrocytes/cc samples showed areas of cartilage separated by areas of remnant fibrin glue. The mass of the samples ranged from 0.07 to 0.12 g at 6 weeks and decreased only slightly by week 12. The glycosaminoglycan content ranged from 2.3 to 9.4 percent for all samples; normal cartilage controls had a content of 7.0 percent. DNA content ranged from 0.63 to 1.4 percent for all samples, with normal pig cartilage having a mean DNA content of 0.285 percent. The samples of fibrin glue alone produced no cartilage, and the chondrocytes alone produced neocartilage samples with a significantly smaller mass (0.47 g at 6 weeks and 0.46 g at 12 weeks) when compared with all samples produced from chondrocytes suspended in fibrin glue (p < 0.03). Gel electrophoreses demonstrated the presence of type II collagen in all sample groups. This study demonstrates that fibrin glue is a suitable polymer for the formation of injectable tissue-engineered cartilage in the nude mouse model. Forty million chondrocytes per cc yielded the best quality cartilage at 6 and 12 weeks when analyzed by histology and content of DNA, glycosaminoglycan, and type II collagen.     

Injectable tissue-engineered cartilage with different chondrocyte sources

Injectable engineered cartilage that maintains a predictable shape and volume would allow recontouring of craniomaxillofacial irregularities with minimally invasive techniques. This study investigated how chondrocytes from different cartilage sources, encapsulated in fibrin polymer, affected construct mass and volume with time. Swine auricular, costal, and articular chondrocytes were isolated and mixed with fibrin polymer (cell concentration of 40 x 10 cells/ml for all groups). Eight samples (1 cm x 1 cm x 0.3 cm) per group were implanted into nude mice for each time period (4, 8, and 12 weeks). The dimensions and mass of each specimen were recorded before implantation and after explantation. Ratios comparing final measurements and original measurements were calculated. Histological, biochemical, and biomechanical analyses were performed. Histological evaluations (n = 3) indicated that new cartilaginous matrix was synthesized by the transplanted chondrocytes in all experimental groups. At 12 weeks, the ratios of dimension and mass (n = 8) for auricular chondrocyte constructs increased by 20 to 30 percent, the ratios for costal chondrocyte constructs were equal to the initial values, and the ratios for articular chondrocyte constructs decreased by 40 to 50 percent. Constructs made with auricular chondrocytes had the highest modulus (n = 3 to 5) and glycosaminoglycan content (n = 4 or 5) and the lowest permeability value (n = 3 to 5) and water content (n = 4 or 5). Constructs made with articular chondrocytes had the lowest modulus and glycosaminoglycan content and the highest permeability value and water content (p < 0.05). The amounts of hydroxyproline (n = 5) and DNA (n = 5) were not significantly different among the experimental groups (p > 0.05). It was possible to engineer injectable cartilage with chondrocytes from different sources, resulting in neocartilage with different properties. Although cartilage made with articular chondrocytes shrank and cartilage made with auricular chondrocytes overgrew, the injectable tissue-engineered cartilage made with costal chondrocytes was stable during the time periods studied. Furthermore, the biomechanical properties of the engineered cartilage made with auricular or costal chondrocytes were superior to those of cartilage made with articular chondrocytes, in this model.     

Effects of cell concentration and growth period on articular and ear chondrocyte transplants for tissue engineering

This study determined the effects of chondrocyte source, cell concentration, and growth period on cartilage production when isolated porcine cells are injected subcutaneously in a nude mouse model. Chondrocytes were isolated from both ear and articular cartilage and were suspended in Ham's F-12 medium at concentrations of 10, 20, 40, and 80 million cells per cubic centimeter. Using the nude mouse model, each concentration group was injected subcutaneously in 100-microl aliquots and was allowed to incubate for 6 weeks in vivo. In addition, cells suspended at a fixed concentration of 40 million cells per cubic centimeter were injected in 100-microl aliquots and were incubated for 1, 2, 3, 4, 5, 6, 9, and 12 weeks. Each concentration or time period studied contained a total of eight mice, with four samples harvested per mouse for a final sample size of 32 constructs. All neocartilage samples were analyzed by histologic characteristics, mass, glycosaminoglycan level, and DNA content. Control groups consisted of native porcine ear and articular cartilage.Specimen mass increased with increasing concentration and incubation time. Ear neocartilage was larger than articular neocartilage at each concentration and time period. At 40 million cells per cubic centimeter, both ear and articular chondrocytes produced optimal neocartilage, without limitations in growth. Specimen mass increased with incubation time up to 6 weeks in both ear and articular samples. No significant variations in glycosaminoglycan content were found in either articular or ear neocartilage, with respect to variable chondrocyte concentration or growth period. Although articular samples demonstrated no significant trends in DNA content over time, ear specimens showed decreasing values through 6 weeks, inversely proportional to increase in specimen mass. Although both articular and ear sources of chondrocytes have been used in past tissue-engineering studies with success, this study indicates that a suspension of ear chondrocytes injected into a subcutaneous location will produce biochemical and histologic data with greater similarity to those of native cartilage. The authors believe that this phenomenon is attributable to the local environment in which isolated chondrocytes from different sources are introduced. The subcutaneous environment of native ear cartilage accommodates subcutaneously injected ear chondrocyte transplants better than articular transplants. Native structural and biochemical cues within the local environment are believed to guide the proliferation of the differentiated chondrocytes.     

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

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

Finite element study of a tissue-engineered cartilage transplant in human tibiofemoral joint

Most tissue-engineered cartilage constructs are more compliant than native articular cartilage (AC) and are poorly integrated to the surrounding tissue. To investigate the effect of an implanted tissue-engineered construct (TEC) with these inferior properties on the mechanical environment of both the engineered and adjacent native tissues, a finite element study was conducted. Biphasic swelling was used to model tibial cartilage and an implanted TEC with the material properties of either native tissue or a decreased elastic modulus and fixed charged density. Creep loading was applied with a rigid impermeable indenter that represented the femur. In comparison with an intact joint, compressive strains in the transplant, surface contact stress in the adjacent native AC and load partitioning between different phases of cartilage were affected by inferior properties of TEC. Results of this study may lead to a better understanding of the complex mechanical environment of an implanted TEC.     

Finite element study of a tissue-engineered cartilage transplant in human tibiofemoral joint

Most tissue-engineered cartilage constructs are more compliant than native articular cartilage (AC) and are poorly integrated to the surrounding tissue. To investigate the effect of an implanted tissue-engineered construct (TEC) with these inferior properties on the mechanical environment of both the engineered and adjacent native tissues, a finite element study was conducted. Biphasic swelling was used to model tibial cartilage and an implanted TEC with the material properties of either native tissue or a decreased elastic modulus and fixed charged density. Creep loading was applied with a rigid impermeable indenter that represented the femur. In comparison with an intact joint, compressive strains in the transplant, surface contact stress in the adjacent native AC and load partitioning between different phases of cartilage were affected by inferior properties of TEC. Results of this study may lead to a better understanding of the complex mechanical environment of an implanted TEC.     

Aerosolization of epidermal cells with fibrin glue for the epithelialization of porcine wounds with unfavorable topography

Aerosolized epidermal cell suspension was previously found to be effective for the epithelialization of full-thickness wounds. This suspension is less expensive than and requires a shorter preparation time than the currently used cultured epithelial autografts. Still, convex and irregular wounds present unfavorable conditions for homogenous dispersion of the aerosolized cell suspension. The authors hypothesized that the addition of fibrin glue to the aerosol of cells would reduce cell movement and ensure homogenous dispersion of the cells, thereby promoting wound epithelialization. The objectives of the study were to evaluate the healing of wounds with unfavorable topography after autotransplantation of an epidermal cell aerosol with and without fibrin glue.Six Yorkshire piglets were studied. An epidermal suspension was made from full-thickness groin skin. Dispase was used to separate the epidermis from the dermis, and trypsin was used to separate the epidermal cells from one another. Twenty-four hours later, full-thickness wounds with unfavorable topography were created adjacent to the vertebral column of six pigs. Twelve wounds were treated with an aerosol of epidermal cell suspension mixed with fibrin glue (study group), and 12 wounds were treated with the same suspension without the fibrin glue (control group). The percentages of total wound contraction and the epithelialized and nonepithelialized areas were evaluated 1, 2, 3, and 4 weeks after aerosolization. The histologic characteristics of the newly formed skin were examined by light microscopy using slides stained with hematoxylin and eosin.Study wounds were characterized by central epithelialization, whereas control wounds were characterized by peripheral epithelialization. Study wounds contracted at a slower rate than control wounds, but wound size was the same in both groups after 4 weeks. The addition of fibrin glue facilitated epithelialization: Study wounds showed 75.5 +/- 22.4 percent (mean +/- SD) and 94.2 +/- 8.8 percent epithelialization after 3 and 4 weeks, respectively, compared with 46.3 +/- 9.5 percent and 47.9 +/- 13.1 percent epithelialization of the control wounds at the same times. These differences between the study and control groups were statistically significant (p < 0.001, paired t test).The addition of fibrin glue to an aerosol of epidermal cells significantly enhances the epithelialization of wounds with unfavorable topography in pigs.     

Experimental use of fibrin glue to induce site-directed osteogenesis from cultured periosteal cells

The purpose of this study was to determine whether a combination of fibrin glue and cultured periosteal cells will result in new bone formation at heterotopic sites in nude mice. Growing cells and developing matrices surrounding periosteal explants from the diaphyses of radii of newborn calves were minced and mixed with fibrin glue in a syringe. The cell/matrix-fibrin glue admixture was then injected into the subcutaneous space on the dorsum of athymic nude mice. After 12 weeks of implantation, gross morphology and histologic investigations showed newly formed bone structures in all cell/matrix-fibrin glue admixtures, but none in fibrin glue injected alone and used as control samples. Osteopontin, a protein important in bone development, was identified by a Western blot assay of the cell/matrix-fibrin glue composite. This study supports the feasibility of initiating site-directed formation of bone structures at heterotopic tissue sites by means of injection of cultured periosteal cells and matrix in a fibrin glue carrier.     

In vitro and in vivo neo-cartilage formation by heterotopic chondrocytes seeded on PGA scaffolds

Implantation of tissue-engineered heterotopic cartilage into joint cartilage defects might be an alternative approach to improve articular cartilage repair. Hence, the aim of this study was to characterize and compare the quality of tissue-engineered cartilage produced with heterotopic (auricular, nasoseptal and articular) chondrocytes seeded on polyglycolic acid (PGA) scaffolds in vitro and in vivo using the nude mice xenograft model. PGA scaffolds were seeded with porcine articular, auricular and nasoseptal chondrocytes using a dynamic culturing procedure. Constructs were pre-cultured 3 weeks in vitro before being implanted subcutaneously in nude mice for 1, 6 or 12 weeks, non-seeded scaffolds were implanted as controls. Heterotopic neo-cartilage quality was assessed using vitality assays, macroscopical and histological scoring systems. Neo-cartilage formation could be observed in vitro in all PGA associated heterotopic chondrocytes cultures and extracellular cartilage matrix (ECM) deposition increased in vivo. The 6 weeks in vivo incubation time point leads to more consistent results for all cartilage species, since at 12 weeks in vivo construct size reductions were higher compared with 6 weeks except for auricular chondrocytes PGA cultures. Some regressive histological changes could be observed in all constructs seeded with all chondrocytes subspecies such as cell-free ECM areas. Particularly, but not exclusively in nasoseptal chondrocytes PGA cultures, ossificated ECM areas appeared. Elastic fibers could not be detected within any neo-cartilage. The neo-cartilage quality did not significantly differ between articular and non-articular chondrocytes constructs. Whether tissue-engineered heterotopic neo-cartilage undergoes sufficient transformation, when implanted into joint cartilage defects requires further investigation.     

碱性成纤维细胞生长因子与胶原对组织工程软骨体外构建的影响

目的：以三维成团培养为培养系统,探讨bFGF与胶原对组织工程软骨体外构建的影响。方法：成团培养兔生长板软骨细胞,设bFGF、胶原及联合作用组。HE染色观察新生组织形态;免疫组化检测Ⅰ、Ⅱ型胶原表达以观察细胞表型;Hoechst 33258法检测细胞DNA含量;羟脯氨酸法与阿新蓝法测定基质中胶原与蛋白多糖的合成。结果：新生软骨的组织学形态近似自然软骨;各实验组软骨细胞DNA含量明显上升;胶原可以显著促进基质的合成;各实验组Ⅰ型胶原的表达少于对照组,Ⅱ型胶原的表达则高于对照组;联合作用组效果更加明显。结论：三维的成团培养可以促进基质合成,有效维持软骨细胞表型;bFGF与胶原有利于工程化软骨构建,其效果具有协同效应,两者联合应用可进一步促进软骨再生。     

Role of pericellular matrix in development of a mechanically functional neocartilage

The role of the chondrocyte pericellular matrix (PCM) was examined in a three-dimensional chondrocyte culture system to determine whether retention of the native pericellular matrix could stimulate collagen and proteoglycan accumulation and also promote the formation of a mechanically functional hyaline-like neocartilage. Porcine chondrocytes and chondrons, consisting of the chondrocyte with its intact pericellular matrix, were maintained in pellet culture for up to 12 weeks. Sulfated glycosaminoclycans and type II collagen were measured biochemically. Immunocytochemistry was used to examine collagen localization as well as cell distribution within the pellets. In addition, the equilibrium compressive moduli of developing pellets were measured to determine whether matrix deposition contributed to the mechanical stiffness of the cartilage constructs. Pellets increased in size and weight over a 6-week period without apparent cell proliferation. Although chondrocytes quickly rebuilt a PCM rich in type VI collagen, chondron pellets accumulated significantly more proteoglycan and type II collagen than did chondrocyte pellets, indicating a greater positive effect of the native PCM. After 5 weeks in chondron pellets, matrix remodeling was evident by microscopy. Cells that had been uniformly distributed throughout the pellets began to cluster between large areas of interterritorial matrix rich in type II collagen. After 12 weeks, clusters were stacked in columns. A rapid increase in compressive strength was observed between 1 and 3 weeks in culture for both chondron and chondrocyte pellets and, by 6 weeks, both had achieved 25% of the equilibrium compressive stiffness of cartilage explants. Retention of the in vivo PCM during chondrocyte isolation promotes the formation of a mechanically functional neocartilage construct, suitable for modeling the responses of articular cartilage to chemical stimuli or mechanical compression.     

Depth-dependent biomechanical and biochemical properties of fetal, newborn, and tissue-engineered articular cartilage

Adult articular cartilage has depth-dependent mechanical and biochemical properties which contribute to zone-specific functions. The compressive moduli of immature cartilage and tissue-engineered cartilage are known to be lower than those of adult cartilage. The objective of this study was to determine if such tissues exhibit depth-dependent compressive properties, and how these depth-varying properties were correlated with cell and matrix composition of the tissue. The compressive moduli of fetal and newborn bovine articular cartilage increased with depth (p<0.05) by a factor of 4-5 from the top 0.1 mm (28+/-13 kPa, 141+/-10 kPa, respectively) to 1 mm deep into the tissue. Likewise, the glycosaminoglycan and collagen content increased with depth (both p<0.001), and correlated with the modulus (both p<0.01). In contrast, tissue-engineered cartilage formed by either layering or mixing cells from the superficial and middle zone of articular cartilage exhibited similarly soft regions at both construct surfaces, as exemplified by large equilibrium strains. The properties of immature cartilage may provide a template for developing tissue-engineered cartilage which aims to repair cartilage defects by recapitulating the natural development and growth processes. These results suggest that while depth-dependent properties may be important to engineer into cartilage constructs, issues other than cell heterogeneity must be addressed to generate such tissues.     

Functional tissue engineering of chondral and osteochondral constructs

Due to the prevalence of osteoarthritis (OA) and damage to articular cartilage, coupled with the poor intrinsic healing capacity of this avascular connective tissue, there is a great demand for an articular cartilage substitute. As the bearing material of diarthrodial joints, articular cartilage has remarkable functional properties that have been difficult to reproduce in tissue-engineered constructs. We have previously demonstrated that by using a functional tissue engineering approach that incorporates mechanical loading into the long-term culture environment, one can enhance the development of mechanical properties in chondrocyte-seeded agarose constructs. As these gel constructs begin to achieve material properties similar to that of the native tissue, however, new challenges arise, including integration of the construct with the underlying native bone. To address this issue, we have developed a technique for producing gel constructs integrated into an underlying bony substrate. These osteochondral constructs develop cartilage-like extracellular matrix and material properties over time in free swelling culture. In this study, as a preliminary to loading such osteochondral constructs, finite element modeling (FEM) was used to predict the spatial and temporal stress, strain, and fluid flow fields within constructs subjected to dynamic deformational loading. The results of these models suggest that while chondral ("gel alone") constructs see a largely homogenous field of mechanical signals, osteochondral ("gel bone") constructs see a largely inhomogeneous distribution of mechanical signals. Such inhomogeneity in the mechanical environment may aid in the development of inhomogeneity in the engineered osteochondral constructs. Together with experimental observations, we anticipate that such modeling efforts will provide direction for our efforts aimed at the optimization of applied physical forces for the functional tissue engineering of an osteochondral articular cartilage substitute.     

Rapid prototyping of anatomically shaped, tissue-engineered implants for restoring congruent articulating surfaces in small joints

Background:  Preliminary studies investigated advanced scaffold design and tissue engineering approaches towards restoring congruent articulating surfaces in small joints.
Materials and methods:  Anatomical femoral and tibial cartilage constructs, fabricated by three-dimensional fibre deposition (3DF) or compression moulding/particulate leaching (CM), were evaluated in vitro and in vivo in an autologous rabbit model. Effects of scaffold pore architecture on rabbit chondrocyte differentiation and mechanical properties were evaluated following in vitro culture and subcutaneous implantation in nude mice. After femoral and tibial osteotomy and autologous implantation of tissue-engineered constructs in rabbit knee joints, implant fixation and joint articulation were evaluated.
Results:  Rapid prototyping of 3DF architectures with 100% interconnecting pores promoted homogeneous distribution of viable cells, glycosaminoglycan (GAG) and collagen type II; significantly greater GAG content and differentiation capacity (GAG/DNA) in vitro compared to CM architectures; and higher mechanical equilibrium modulus and dynamic stiffness (at 0.1 Hz). Six weeks after implantation, femoral and tibial constructs had integrated with rabbit bone and knee flexion/extension and partial load bearing were regained. Histology demonstrated articulating surfaces between femoral and tibial constructs for CM and 3DF architectures; however, repair tissue appeared fibrocartilage-like and did not resemble implanted cartilage.
Conclusions:  Anatomically shaped, tissue-engineered constructs with designed mechanical properties and internal pore architectures may offer alternatives for reconstruction or restoration of congruent articulating surfaces in small joints.     

Oxygen gradients in tissue-engineered PEGT/PBT cartilaginous constructs: measurement and modeling

The supply of oxygen within three-dimensional tissue-engineered (TE) cartilage polymer constructs is mainly by diffusion. Oxygen consumption by cells results in gradients in the oxygen concentration. The aims of this study were, firstly, to identify the gradients within TE cartilage polymer constructs and, secondly, to predict the profiles during in vitro culture. A glass microelectrode system was adapted and used to penetrate cartilage and TE cartilaginous constructs, yielding reproducible measurements with high spatial resolution. Cartilage polymer constructs were cultured for up to 41 days in vitro. Oxygen concentrations, as low as 2-5%, were measured within the center of these constructs. At the beginning of in vitro culture, the oxygen gradients were steeper in TE constructs in comparison to native tissue. Nevertheless, during the course of culture, oxygen concentrations approached the values measured in native tissue. A mathematical model was developed which yields oxygen profiles within cartilage explants and TE constructs. Model input parameters were assessed, including the diffusion coefficient of cartilage (2.2 x 10(-9)) + (0.4 x 10(-9) m(2) s(-1)), 70% of the diffusion coefficient of water and the diffusion coefficient of constructs (3.8 x 10(-10) m(2) s(-1)). The model confirmed that chondrocytes in polymer constructs cultured for 27 days have low oxygen requirements (0.8 x 10(-19) mol m(-3) s(-1)), even lower than chondrocytes in native cartilage. The ability to measure and predict local oxygen tensions offers new opportunities to obtain more insight in the relation between oxygen tension and chondrogenesis.     

Chondrocyte suspension in fibrin glue

Autologous chondrocyte implantation has been shown to be a promising method for treatment of deep articular cartilage defects. The hyaline cartilage formed by implanted autologous chondrocytes has biomechanical properties similar to those of natural articular cartilage. Between June 2006 and September 2008 we performed Autologous chondrocyte implantation (ACI) in 50 patients and the chondrocytes were supported in fibrin glue. The cartilage biopsy samples were taken from the non-weight bearing area of the patient’s femoral condyle and the samples were transferred to the cell culture laboratory. Chondrocyte were kept in culture about 20 days. Fibrin glue was used as a three dimensional carrier for chondrocyte implantation. A 450 ml of patient’s own blood was collected prior to transplantation to produce autologous fibrinogen. Alternatively the allogenic fibrinogen was prepared from Regional Blood Center voluntary donors. Before surgery the chondrocyte suspension was mixed with fibrin glue and gel—like fibrograft was prepared. The total number of cells and the size of fibrograft depended on the defect size in the knee. Our results suggest that ACI technique with fibrin glue is a promising method for treatment of cartilage defect.     

Evaluation of an in vivo heterotopic model of osteogenic differentiation of equine bone marrow and muscle mesenchymal stem cells in fibrin glue scaffold

Autologous mesenchymal stem cells (MSCs) have been used as a potential cell-based therapy in various animal and human diseases. Their differentiation capacity makes them useful as a novel strategy in the treatment of tissue injury in which the healing process is compromised or delayed. In horses, bone healing is slow, taking a minimum of 6–12 months. The osteogenic capacity of equine bone marrow and muscle MSCs mixed with fibrin glue or phosphate-buffered saline (PBS) as a scaffold is assessed. Bone production by the following groups was compared: Group 1, bone marrow (BM) MSCs in fibrin glue; Group 2, muscle (M) MSCs in fibrin glue; Group 3, BM MSCs in PBS; Group 4, M MSCs in PBS and as a control; Group 5, fibrin glue without cells. BM and M MSCs underwent osteogenic stimulation for 48 h prior to being injected intramuscularly into nude mice. After 4 weeks, the mice were killed and muscle samples were collected and evaluated for bone formation and mineralization by using radiology, histochemistry and immunohistochemistry. Positive bone formation and mineralization were confirmed in Group 1 in nude mice based on calcium deposition and the presence of osteocalcin and collagen type I; in addition, a radiopaque area was observed on radiographs. However, no evidence of mineralization or bone formation was observed in Groups 2–5. In this animal model, equine BM MSCs mixed with fibrin glue showed better osteogenic differentiation capacity compared with BM MSCs in PBS and M MSCs in either carrier.     

软骨基质来源定向结构支架复合软骨细胞体外构建组织工程软骨的研究

目的：探讨采用软骨细胞外基质材料制备的定向结构软骨支架复合软骨细胞,在体外静态培养条件下生成组织工程软骨的可能性。方法：制备牛关节软骨细胞外基质材料,利用温度梯度热诱导相分离技术构建具备垂直定向孔道结构的软骨支架,同时采用传统冷冻干燥方法制备非定向支架,检测两组支架的力学性能;提取兔关节软骨细胞,分别接种两组支架,体外静态培养2周及4周后取材,对构建的组织工程软骨进行组织切片染色、生物化学分析及生物力学检测。结果：定向软骨支架的压缩弹性模量数值明显高于非定向软骨支架,体外培养时定向支架上种子细胞在3-9d内增殖高于非定向支架,差异有统计学意义（P〈0．05）;体外静态培养4周后形成的两组新生组织工程软骨进行软骨特异性染色均呈阳性,在定向组新生软骨切片中在垂直方向上可见大量呈规则平行排列的粗大胶原纤维,两组新生软骨的生物化学检测包括总DNA、总GAG及总胶原含量差异无统计学意义（P〉0．05）。定向组织工程软骨压缩弹性模量在2周及4周时均高于非定向组织工程软骨,差异有统计学意义（P〈0．05）。但两组组织工程软骨上述指标均显著低于正常关节软骨（P〈0．05）。结论：软骨细胞外基质材料制备的定向结构软骨支架复合软骨细胞,在体外静态培养条件下能够成功生成具有定向纤维结构的组织工程软骨,并可以有效促进新生软骨组织力学性能的提升,在软骨组织工程中具有良好的应用前景。     

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.     

Induced Collagen Cross-Links Enhance Cartilage Integration

Articular cartilage does not integrate due primarily to a scarcity of cross-links and viable cells at the interface. The objective of this study was to test the hypothesis that lysyl-oxidase, a metalloenzyme that forms collagen cross-links, would be effective in improving integration between native-to-native, as well as tissue engineered-to-native cartilage surfaces. To examine these hypotheses, engineered cartilage constructs, synthesized via the self-assembling process, as well as native cartilage, were implanted into native cartilage rings and treated with lysyl-oxidase for varying amounts of time. For both groups, lysyl-oxidase application resulted in greater apparent stiffness across the cartilage interface 2–2.2 times greater than control. The construct-to-native lysyl-oxidase group also exhibited a statistically significant increase in the apparent strength, here defined as the highest observed peak stress during tensile testing. Histology indicated a narrowing gap at the cartilage interface in lysyl-oxidase treated groups, though this alone is not sufficient to indicate annealing. However, when the morphological and mechanical data are taken together, the longer the duration of lysyl-oxidase treatment, the more integrated the interface appeared. Though further data are needed to confirm the mechanism of action, the enhancement of integration may be due to lysyl-oxidase-induced pyridinoline cross-links. This study demonstrates that lysyl-oxidase is a potent agent for enhancing integration between both native-to-native and native-to-engineered cartilages. The fact that interfacial strength increased manifold suggests that cross-linking agents should play a significant role in solving the difficult problem of cartilage integration. Future studies must examine dose, dosing regimen, and cellular responses to lysyl-oxidase to optimize its application.