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.     

The use of fibrin glue in skin grafts and tissue-engineered skin replacements: a review.

Fibrin glue has been widely used as an adhesive in plastic and reconstructive surgery. This article reviews the advantages and disadvantages of its use with skin grafts and tissue-engineered skin substitutes. Fibrin glue has been shown to improve the percentage of skin graft take, especially when associated with difficult grafting sites or sites associated with unavoidable movement. Evidence also suggests improved hemostasis and a protective effect resulting in reduced bacterial infection. Fibrin, associated with fibronectin, has been shown to support keratinocyte and fibroblast growth both in vitro and in vivo, and may enhance cellular motility in the wound. When used as a delivery system for cultured keratinocytes and fibroblasts, fibrin glue may provide similar advantages to those proven with conventional skin grafts. Fibrin glue has also been shown to be a suitable delivery vehicle for exogenous growth factors that may in the future be used to accelerate wound healing.     

Successful transplantation of three tissue-engineered cell types using capsule induction technique and fibrin glue as a delivery vehicle

Recent advances in cell biology and tissue engineering have used various delivery vehicles for transplanting varying cell cultures with limited success. These techniques are frequently complicated by tissue necrosis, infection, and resorption. The purpose of this study was to investigate whether urothelium cells, tracheal epithelial cells, and preadipocytes cultured in vitro could be successfully transplanted onto a prefabricated capsule surface by using fibrin glue as a delivery vehicle, with the ultimate goal for use in reconstruction. In the first step of the animal study, tissue specimens (bladder urothelium, tracheal epithelial cells, epididymal fat pad) were harvested for in vitro cell culturing, and a silicone block was implanted subcutaneously or within the anterior rectus sheath to induce capsule formation. After 6 to 10 days, when primary cultures were confluent, the animals were re-anesthetized, the newly formed capsule pouches were incised, and the suspensions of cultured urothelia cells (n = 40), tracheal epithelial cells (n = 32), and preadipocytes (n = 40) were implanted onto the capsule surface in two groups, one using standard culture medium as a delivery vehicle and the second using fibrin glue. Histologic sections were taken, and different histomorphologic studies were performed according to tissue type. Consistently in all animals, a highly vascularized capsule was induced by the silicon material. In all animals in which the authors used fibrin glue as a delivery vehicle, they could demonstrate a successful reimplantation of cultured urothelium cells, tracheal epithelial cells, or preadipocytes. Their animal studies showed that capsule induction in combination with fibrin glue as a delivery vehicle is a successful model for transplantation of different in vivo cultured tissue types.     

Construction of tissue-engineered cartilage using human placenta-derived stem cells

Human placenta-derived stem cells (hPDSCs) were isolated by trypsinization and further induced into cartilage cells in vitro. The engineered cartilage was constructed by combining hPDSCs with collagen sponge and the cartilage formation was observed by implantation into nude mice. Results showed that hPDSCs featured mesenchymal stem cells and maintained proliferation in vitro for over 30 passages while remaining undifferentiated. All results indicated that hPDSCs have the potential to differentiate into functional cartilage cells in vitro when combined with collagen sponge, which provided experimental evidence for prospective clinical application.     

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.     

Experimental and clinical applications of fibrin glue

A 2-year experience with laboratory and clinical applications of fibrin glue is presented. An autologous technique, which eliminates the danger of multidonor preparations, has been developed in our blood bank. While one can obtain different fibrinogen concentrations from the same amount of a patient's blood, in vitro mechanical testing demonstrated that at higher fibrinogen concentrations there is an increase in shear adhesive strength. Evaluation of skin-graft take in 16 Sprague-Dawley rats did not demonstrate significant differences in healing when adhesive use was compared with suture technique. In a clinical study, four different groups of patients (facial burns, hand burns, difficult graft sites, and miscellaneous surgical applications) benefited from autologous or single-donor fibrin glue for a total of 82 cases. There are several distinct advantages to the use of fibrin adhesive: The autologous technique eliminates the risk of transmissible viral diseases (AIDS, hepatitis); it can be used as a sealant in the treatment of seromas, dural leaks, and lymphoceles; and it improves hemostasis and early graft adherence. Face and hands are resurfaced with sheet grafts in a single procedure, obtaining a better aesthetic result with complete graft take and immediate start of physical therapy. Neither sutures nor pressure dressings are required. The minimal postoperative care associated with early return to normal activities seems to increase the satisfaction of patients and nurse personnel.     

Adhesion of tissue-engineered cartilate to native cartilage

Reconstruction of cartilaginous defects to correct both craniofacial deformities and joint surface irregularities remains a challenging and controversial clinical problem. It has been shown that tissue-engineered cartilage can be produced in a nude mouse model. Before tissue-engineered cartilage is used clinically to fill in joint defects or to reconstruct auricular or nasal cartilaginous defects, it is important to determine whether it will integrate with or adhere to the adjacent native cartilage at the recipient site. The purpose of this study was to determine whether tissue-engineered cartilage would adhere to adjacent cartilage in vivo. Tissue-engineered cartilage was produced using a fibrin glue polymer (80 mg/cc purified porcine fibrinogen polymerized with 50 U/cc bovine thrombin) mixed with fresh swine articular chondrocytes. The polymer/chondrocyte mixture was sandwiched between two 6-mm-diameter discs of fresh articular cartilage. These constructs were surgically inserted into a subcutaneous pocket on the backs of nude mice (n = 15). The constructs were harvested 6 weeks later and assessed histologically, biomechanically, and by electron microscopy. Control samples consisted of cartilage discs held together by fibrin glue alone (no chondrocytes) (n = 10). Histologic evaluation of the experimental constructs revealed a layer of neocartilage between the two native cartilage discs. The neocartilage appeared to fill all irregularities along the surface of the cartilage discs. Safranin-O and toluidine blue staining indicated the presence of glycosaminoglycans and collagen, respectively. Control samples showed no evidence of neocartilage formation. Electron microscopy of the neocartilage revealed the formation of collagen fibers similar in appearance to the normal cartilage matrix in the adjacent native cartilage discs. The interface between the neocartilage and the native cartilage demonstrated neocartilage matrix directly adjacent to the normal cartilage matrix without any gaps or intervening capsule. The mechanical properties of the experimental constructs, as calculated from stress-strain curves, differed significantly from those of the control samples. The mean modulus for the experimental group was 0.74 +/- 0.22 MPa, which was 3.5 times greater than that of the control group (p < 0.0002). The mean tensile strength of the experimental group was 0.064 +/- 0.024 MPa, which was 62.6 times greater than that of the control group (p < 0.0002). The mean failure strain of the experimental group was 0.16 +/- 0.061 percent, which was 4.3 times greater than that of the control group (p < 0.0002). Finally, the mean fracture energy of the experimental group was 0.00049 +/- 0.00032 J, which was 15.6 times greater than that of the control group. Failure occurred in all cases at the interface between neocartilage and native cartilage. This study demonstrated that tissue-engineered cartilage produced using a fibrin-based polymer does adhere to adjacent native cartilage and can be used to join two separate pieces of cartilage in the nude mouse model. Cartilage pieces joined in this way can withstand forces significantly greater than those tolerated by cartilage samplesjoined only by fibrin glue.     

Effects of mixing intensity on tissue-engineered cartilage

Mechanical forces regulate the structure and function of many tissues in vivo; recent results indicate that the mechanical environment can decisively influence the development of engineered tissues cultured in vitro. To investigate the effects of the hydrodynamic environment on tissue-engineered cartilage, primary bovine calf chondrocytes were seeded on fibrous polyglycolic acid meshes and cultured in spinner flasks either statically or at one of nine different turbulent mixing intensities. In medium from unmixed flasks, CO(2) accumulated and O(2) was depleted, whereas in medium from mixed flasks the concentrations of both gases approached their equilibrium values. Relative to constructs exposed to nonmixed conditions, constructs exposed to mixing contained higher fractions of collagen, synthesized and released more GAG, but contained lower fractions of GAG. Across the wide range of mixing intensities investigated, the presence or absence of mixing, but not the intensity of the mixing, was the primary determinant of the GAG and collagen content in the constructs. The all-or-none nature of these responses may provide insight into the mechanism(s) by which engineered cartilage perceives changes in its hydrodynamic environment and responds by modifying extracellular matrix production and release. 2001 John Wiley & Sons, Inc.     

以可注射性纤维蛋白封闭剂为支架体内构建组织工程软骨

目的: 研究以纤维蛋白封闭剂（FS）为载体复合人胚关节软骨细胞体内构建可注射性组织工程软骨的可行性。方法:常规分离消化,体外单层培养胎儿关节软骨细胞,观察软骨细胞的生物学特性。分别将1×107、2×107、3×107第4代软骨细胞与FS混合接种于裸鼠皮下, 并于第10周取材判断体内形成软骨的能力。结果: 3~4代软骨细胞保持了很高的增殖和分泌基质的能力。软骨细胞与FS的复合物体内接种后各组均可形成软骨样组织块,其湿重、GAG含量随着接种细胞数量的增多而增高,各组之间差异具有显著性（p<0.05）。3×107细胞组GAG含量与正常人胚关节软骨没有差异（p>0.05）。组织切片显示软骨细胞位于类似正常软骨组织的陷窝中,阿尔新蓝染色及II型胶原表达阳性,细胞内富含高尔基体、粗面内质网及大量分泌泡。结论: FS和人胚关节软骨细胞可以作为理想的支架材料和种子细胞应用于可注射软骨组织的构建。     

Scaffoldless tissue-engineered cartilage for studying transforming growth factor beta-mediated cartilage formation

Reduced transforming growth factor beta (TGF-β) signaling is associated with osteoarthritis (OA). TGF-β is thought to act as a chondroprotective agent and provide anabolic cues to cartilage, thus acting as an OA suppressor in young, healthy cartilage. A potential approach for treating OA is to identify the factors that act downstream of TGF-β's anabolic pathway and target those factors to promote cartilage regeneration or repair. The aims of the present study were to (a) develop a scaffoldless tissue-engineered cartilage model with reduced TGF-β signaling and disrupted cartilage formation and (b) validate the system for identifying the downstream effectors of TGF-β that promote cartilage formation. Sox9 was used to validate the model because Sox9 is known to promote cartilage formation and TGF-β regulates Sox9 activity. Primary bovine articular chondrocytes were grown in Transwell supports to form cartilage tissues. An Alk5/TGF-β type I receptor inhibitor, SB431542, was used to attenuate TGF-β signaling, and an adenovirus encoding FLAG-Sox9 was used to drive the expression of Sox9 in the in vitro-generated cartilage. SB431542-treated tissues exhibited reduced cartilage formation including reduced thicknesses and reduced proteoglycan staining compared with control tissue. Expression of FLAG-Sox9 in SB431542-treated cartilage allowed the formation of cartilage despite antagonism of the TGF-β receptor. In summary, we developed a three-dimensional in vitro cartilage model with attenuated TGF-β signaling. Sox9 was used to validate the model for identification of anabolic agents that counteract loss of TGF-β signaling. This model has the potential to identify additional anabolic factors that could be used to repair or regenerate damaged cartilage.     

Use of fibrin glue in sucrose gap method

Isolated frog nerves or rabbit sinus node strips were mounted in a single sucrose gap chamber. A fibrin glue Tissucol was used in this arrangement as an intercompartment seal. Under such conditions, the specimens produced stable trans-gap action potentials of relatively high amplitude. In other experiments the gap was filled with fibrin in place of sucrose solution. The results obtained in the fibrin gap experiments resembled these mentioned above.     

Salmon fibrin glue in rats: Antibody studies

Fibrin sealants and topical thrombin preparations are often used for haemostatic and sealing applications in clinical practice. Some of these preparations contain coagulation factors from bovine sources. To minimize the risk of infection and immunogenicity connected with mammalian blood products, proteins derived from the plasma of farmed Atlantic salmon have been considered as an alternative to these mammalian sources. The purpose of this study is to characterize the immunogenicity of salmon fibrin glue in an animal model focusing on crossreactivity of IgG antibodies to host endogenous counterparts. After two immunizations with salmon fibrin glue, rats developed antibodies of IgG and IgM type to both fibrin glue components. Weak crossreactivity to endogenous fibrinogen and thrombin was seen in a subset of rats after the second application of salmon proteins. Coagulation tests showed that salmon fibrin application has no effect on coagulation profiles in mammalian hosts, consistent with previous reports that found no evidence of significant crossreactivity with host proteins. These studies support the potential suitability of salmon fibrin glue for the development of preparations with clinical impact. Before human use can be considered, however, additional data about safety of this preparation in other animal models, including large animal studies, should be obtained.