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.     

Nucleus pulposus repair with cultured autologous elastic cartilage derived chondrocytes

Low back pain is one of the most common medical conditions in the Western world. Disc degeneration, an inevitable process of ageing, is one of the major causes of low back pain. Autologous chondrocyte transplantation (ACT) is an increasingly popular method of addressing pathological disorders of cartilage. The purpose of our study was to determine whether autologous chondrocytes from elastic cartilage could survive and synthesise a cartilage specific matrix in the intervertebral disc of rabbits. Sixteen lumbar intervertebral discs (IVD) of New Zealand White rabbits were analysed. In 6 IVD, the nucleus pulposus was evacuated and replaced with tissue engineered autologous chondrocytes from auricular cartilage. In the second group, only the nucleus pulposus was evacuated from 6 IVD, with no chondrocytes implantation. Four non-operated IVD were used as a control. Six months after the operation, the animals were euthanized and the IVD were analysed histologically. Autologous cartilage implants were well tolerated by the host for up to six months in vivo. There was only hyaline-like cartilage in the place of the nucleus pulposus. We could not detect any elastic fibres in the new cartilage matrix. In IVD from which only the nucleus pulposus was evacuated and no chondrocytes were implanted, just fibrous tissue was found instead of nucleus pulposus. The overall histological analysis of new cartilage produced after implantation in our study confirmed the hypothesis that ACT from auricular cartilage can be implanted into the IVD instead of the nucleus pulposus and that a significant percentage of implanted chondrocytes survive and produce hyaline-like cartilage.     

Growth Factor Stimulation Improves the Structure and Properties of Scaffold-Free Engineered Auricular Cartilage Constructs

The reconstruction of the external ear to correct congenital deformities or repair following trauma remains a significant challenge in reconstructive surgery. Previously, we have developed a novel approach to create scaffold-free, tissue engineering elastic cartilage constructs directly from a small population of donor cells. Although the developed constructs appeared to adopt the structural appearance of native auricular cartilage, the constructs displayed limited expression and poor localization of elastin. In the present study, the effect of growth factor supplementation (insulin, IGF-1, or TGF-β1) was investigated to stimulate elastogenesis as well as to improve overall tissue formation. Using rabbit auricular chondrocytes, bioreactor-cultivated constructs supplemented with either insulin or IGF-1 displayed increased deposition of cartilaginous ECM, improved mechanical properties, and thicknesses comparable to native auricular cartilage after 4 weeks of growth. Similarly, growth factor supplementation resulted in increased expression and improved localization of elastin, primarily restricted within the cartilaginous region of the tissue construct. Additional studies were conducted to determine whether scaffold-free engineered auricular cartilage constructs could be developed in the 3D shape of the external ear. Isolated auricular chondrocytes were grown in rapid-prototyped tissue culture molds with additional insulin or IGF-1 supplementation during bioreactor cultivation. Using this approach, the developed tissue constructs were flexible and had a 3D shape in very good agreement to the culture mold (average error <400 µm). While scaffold-free, engineered auricular cartilage constructs can be created with both the appropriate tissue structure and 3D shape of the external ear, future studies will be aimed assessing potential changes in construct shape and properties after subcutaneous implantation.     

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.     

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.     

Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo.

Articular cartilage exhibits little intrinsic repair capacity, and new tissue engineering approaches are being developed to promote cartilage regeneration using cellular therapies. The goal of this study was to examine the chondrogenic potential of adipose tissue-derived stromal cells. Stromal cells were isolated from human subcutaneous adipose tissue obtained by liposuction and were expanded and grown in vitro with or without chondrogenic media in alginate culture. Adipose-derived stromal cells abundantly synthesized cartilage matrix molecules including collagen type II, VI, and chondroitin 4-sulfate. Alginate cell constructs grown in chondrogenic media for 2 weeks in vitro were then implanted subcutaneously in nude mice for 4 and 12 weeks. Immunohistochemical analysis of these samples showed significant production of cartilage matrix molecules. These findings document the ability of adipose tissue-derived stromal cells to produce characteristic cartilage matrix molecules in both in vitro and in vivo models, and suggest the potential of these cells in cartilage tissue engineering.     

The role of bioreactors in cartilage tissue engineering

Cartilage tissue engineering is concerned with developing in vitro cartilage implants that closely match the properties of native cartilage, for eventual implantation to replace damaged cartilage. The three components to cartilage tissue engineering are cell source, such as in vitro expanded autologous chondrocytes or mesenchymal progenitor cells, a scaffold onto which the cells are seeded and a bioreactor which attempts to recreate the in vivo physicochemical conditions in which cartilage develops. Although much progress has been made towards the goal of developing clinically useful cartilage constructs, current constructs have inferior physicochemical properties than native cartilage. One of the reasons for this is the neglect of mechanical forces in cartilage culture. Bioreactors have been defined as devices in which biological or biochemical processes can be re-enacted under controlled conditions e.g. pH, temperature, nutrient supply, O2 tension and waste removal. The purpose of this review is to detail the role of bioreactors in the engineering of cartilage, including a discussion of bioreactor designs, current state of the art and future perspectives.     

Ectopic osteogenesis and chondrogenesis of bone marrow stromal stem cells in alginate system

In orthopedics, the regeneration and repair of cartilage or bone defects after trauma, cancer, or metabolic disorders is still a major clinical challenge. Through developmental plasticity, bone marrow mesenchymal stem cells (BMSSCs) are important seed cells for the musculoskeletal tissue engineering approach. The present study sought to determine the ectopic osteogenic and chondrogenic ability of BMSSCs in combination with a scaffolding material made from alginate gel. After isolation from the bone marrow of BALB/C mice, BMSSCs were expanded in vitro and induced to chondrogenesis or osteogenesis for 14 days, respectively. Subsequently, these induced cells were seeded into alginate gel, and the constructs implanted into BALB/C nude mice subcutaneously for up to 8 weeks. In the histological analysis, the transmission electron microscopy of the retrieved specimens at various intervals showed obvious trends of ectopic cartilage or bone formation along with the alteration of the cellular phenotype. Simultaneously, the results of the immunohistochemical staining and RT-PCR both confirmed the expression of specific extracellular matrix (ECM) markers for cartilaginous tissue, such as collagen type II (Col-II), SOX9, and aggrecan, or alternatively, markers for osteoid tissue, such as osteopontin (OPN), osteocalcin (OCN), and collagen type I (Col-I). During subcutaneous implantation, the elevating production of ECM and the initiation of the characteristic structure were closely correlated with the increase of time. In contrast, there was an apparent degradation and resorption of the scaffolding material in blank controls, but with no newly formed tissues. Finally, the constructs that were made of non-induced BMSSCs nearly disappeared during the 8 weeks after implantation. Therefore, it is suggested that alginate gel, which is combined with BMSSCs undergoing differentiation into skeletal lineages, may represent a useful strategy for the clinical reconstruction of bone and cartilage defects.     

Transdermal photopolymerization of poly(ethylene oxide)-based injectable hydrogels for tissue-engineered cartilage

Transdermal photopolymerization, a minimally invasive method for implantation, was used to subcutaneously place a mixture of polymer and isolated chondrocytes to regenerate cartilage tissue in vivo. Semi-interpenetrating networks of varying proportions of poly(ethylene oxide)-dimethacrylate and poly(ethylene oxide) and primary bovine articular chondrocytes were implanted in athymic mice. Four mice (12 implants) were harvested at 2, 4, and 7 weeks. Chondrocytes survived implantation and photopolymerization and formed neocartilage containing 1.5 to 2.9% wet weight collagen and 4 to 7% glycosaminoglycan. Thirty-five percent of the total collagen was type II collagen. Histologic analysis exhibited tissue structure resembling neocartilage, and safranin O staining demonstrated glycosaminoglycan distribution throughout the hydrogels. This study demonstrates the potential use of transdermal photopolymerization for minimally invasive subcutaneous implantation of hydrogels and chondrocytes for in vivo cartilage regeneration.     

Prefabrication of 3D Cartilage Contructs: Towards a Tissue Engineered Auricle – A Model Tested in Rabbits

The reconstruction of an auricle for congenital deformity or following trauma remains one of the greatest challenges in reconstructive surgery. Tissue-engineered (TE) three-dimensional (3D) cartilage constructs have proven to be a promising option, but problems remain with regard to cell vitality in large cell constructs. The supply of nutrients and oxygen is limited because cultured cartilage is not vascular integrated due to missing perichondrium. The consequence is necrosis and thus a loss of form stability. The micro-surgical implantation of an arteriovenous loop represents a reliable technology for neovascularization, and thus vascular integration, of three-dimensional (3D) cultivated cell constructs. Auricular cartilage biopsies were obtained from 15 rabbits and seeded in 3D scaffolds made from polycaprolactone-based polyurethane in the shape and size of a human auricle. These cartilage cell constructs were implanted subcutaneously into a skin flap (15×8 cm) and neovascularized by means of vascular loops implanted micro-surgically. They were then totally enhanced as 3D tissue and freely re-implanted in-situ through microsurgery. Neovascularization in the prefabricated flap and cultured cartilage construct was analyzed by microangiography. After explantation, the specimens were examined by histological and immunohistochemical methods. Cultivated 3D cartilage cell constructs with implanted vascular pedicle promoted the formation of engineered cartilaginous tissue within the scaffold in vivo. The auricles contained cartilage-specific extracellular matrix (ECM) components, such as GAGs and collagen even in the center oft the constructs. In contrast, in cultivated 3D cartilage cell constructs without vascular pedicle, ECM distribution was only detectable on the surface compared to constructs with vascular pedicle. We demonstrated, that the 3D flaps could be freely transplanted. On a microangiographic level it was evident that all the skin flaps and the implanted cultivated constructs were well neovascularized. The presented method is suggested as a promising alternative towards clinical application of engineered cartilaginous tissue for plastic and reconstructive surgery.     

A second-generation autologous chondrocyte implantation approach to the treatment of focal articular cartilage defects

Autologous chondrocyte implantation (ACI) is the most widely used cell-based surgical procedure for the repair of articular cartilage defects. Challenges to successful ACI outcomes include limitation in defect size and geometry as well as inefficient cell retention. Second-generation ACI procedures have thus focused on developing three-dimensional constructs using native and synthetic biomaterials. Clinically significant and satisfactory results from applying autologous chondrocytes seeded in fibrin within a biodegradable polymeric material were recently reported. In the future, third-generation cell-based articular cartilage repair should focus on the use of chondroprogenitor cells and biofunctionalized biomaterials for more extensive and permanent repair.     

Engineering of osteoinductive grafts by isolation and expansion of ovine bone marrow stromal cells directly on 3D ceramic scaffolds

In this work, we investigated whether osteoinductive constructs can be generated by isolation and expansion of sheep bone marrow stromal cells (BMSC) directly within three-dimensional (3D) ceramic scaffolds, bypassing the typical phase of monolayer (2D) expansion prior to scaffold loading. Nucleated cells from sheep bone marrow aspirate were seeded into 3D ceramic scaffolds either by static loading or under perfusion flow and maintained in culture for up to 14 days. The resulting constructs were exposed to enzymatic treatment to assess the number and lineage of extracted cells, or implanted subcutaneously in nude mice to test their capacity to induce bone formation. As a control, BMSC expanded in monolayer for 14 days were also seeded into the scaffolds and implanted. BMSC could be isolated and expanded directly in the 3D ceramic scaffolds, although they proliferated slower than in 2D. Upon ectopic implantation, the resulting constructs formed a higher amount of bone tissue than constructs loaded with the same number of 2D-expanded cells. Constructs cultivated for 14 days generated significantly more bone tissue than those cultured for 3 days. No differences in bone formation were found between samples seeded by static loading or under perfusion. In conclusion, the culture of bone marrow nucleated cells directly on 3D ceramic scaffolds represents a promising approach to expand BMSC and streamline the engineering of osteoinductive grafts.     

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.     

Engineering of bone tissue with porcine bone marrow stem cells in three-dimensional trabecular metal: in vitro and in vivo studies

The aim of this study was to investigate capability of cell attachment and ectopic bone formation in pigs after either ex vivo transplantation and expansion of bone marrow stem cells (BMSc) into three-dimensional porous tantalum, or porous tantalum supplemented with BMSc. After 24 hours incubation, cells adhering to the porous tantalum discs were quantified by means of scintillation counting of 3H-thymidine-labeled cells. After 7 days of incubation, the cell-loaded porous tantalum discs were harvested for histological analysis or implanted in the infrasternal muscle; an empty disc and disc implanted immediately after cell loading served as controls. All implants were taken out after 8 weeks of implantation and histological examination was performed. The results of in vitro cell attachment to the porous tantalum discs were not improved significantly with gelatin, collagen or fibronectin coatings. Histological analysis of cell loaded discs in vitro demonstrated viable BMSc within the 3-D tantalum structure. In vivo bone induction was demonstrated when the porous tantalum discs were cultured with BMSc. Our findings indicated that porous tantalum was suitable for cell attachment, and ectopic bone formation in pigs was achieved by means of BMSc cultured with porous tantalum. The present study suggests that cell-mediated hard bone tissue repair technology makes it possible to prefabricate autologous BMSc into three-dimensional trabecular metal in order to engineer bone tissue.     

Generation of a scaffold free cartilage-like implant from a small amount of starting material

INTRODUCTION: An autologous cellular based treatment of a traumatic cartilage injury requires a procedure whereby a biopsy of healthy cartilage is removed from the patient and the cells isolated and expanded by monolayer passage. This increases the cell number to required levels but also leads to a de-differentiation of the cells. We aim to produce a scaffold-free, de-novo implant from a biopsy of cartilage. METHODS: Bovine chondrocytes were isolated from a small biopsy and expanded. The chondrocytic phenotype of the monolayer expanded cells was recovered during a period of culture in alginate and the effect of factors such as IGF1, TFGbeta1 and dexamethasone was investigated. RESULTS: During the alginate culture period a pre-treatment with IGF1 and dexamethasone was shown to have little effect. IGF1 however increased the glycosaminoglycan/DNA (GAG/DNA) content on day 14 to 84.95+/-5 ng/ng compared with 37.3+/-1.8 ng/ng in the controls (P<0.001). 35S labeling demonstrated an increased GAG synthesis in the presence of IGF1 (P<0.001). IGF1 also induced a increase of DNA content 1383+/-314 ng/bead compared to 512+/-19 ng/bead in the controls (P<0.001).The cells were released from the alginate and cultured in a silicon mould for a further 14 days to obtain a three dimensional implant. Releasing the cells from the alginate and casting in a mould produced an implant of defined shape which contained no foreign material. After 31 days of culture the implants contained 152.4+/-13.14 ng/ng GAG/DNA and 42.93+/-10.23 ng/ng collagen II. DISCUSSION: We believe alginate released chondrocytes provide a real alternative to artificial scaffolds.     

Monoclonal antibody 11-fibrau: a useful marker to characterize chondrocyte differentiation stage

The aim of this study was to determine the feasibility of discriminating between differentiated and dedifferentiated chondrocytes by using the Mab 11-fibrau. Mab 11-fibrau did not bind to differentiated chondrocytes in cartilage of human knee joint, auricle, or nasal septum. During monolayer culture, when cells dedifferentiate, the number of 11-fibrau positive cells gradually increased and reached up to 100% after 4 passages. When differentiated chondrocytes were cultured in alginate, most (90--95%) of the cells remained 11-fibrau negative, in accordance with previous studies demonstrating that differentiated chondrocytes cultured in alginate keep their phenotype. Dedifferentiated (11-fibrau positive) cells were subjected to different redifferentiation regimes. As a well-known fact, cultures in alginate in medium where FCS was replaced by IGF1 and TGF beta 2 results in increased collagen type II formation, indicative for redifferentiation. However, the cells remained 11-fibrau positive, suggesting they are not (yet) fully redifferentiated. On the other hand, when dedifferentiated cells (after 4 passages in monolayer culture) were seeded in a biomaterial and implanted subcutaneously in a nude mouse, the newly formed cartilage matrix contained collagen type II and the 11-fibrau staining on the cells had disappeared. Our results indicate that 11-fibrau may be a reliable and sensitive marker of chondrocyte phenotype.     

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.     

Considerations on the use of ear chondrocytes as donor chondrocytes for cartilage tissue engineering

Articular cartilage is often used for research on cartilage tissue engineering. However, ear cartilage is easier to harvest, with less donor-site morbidity. The aim of this study was to evaluate whether adult human ear chondrocytes were capable of producing cartilage after expansion in monolayer culture. Cell yield per gram of cartilage was twice as high for ear than for articular cartilage. Moreover, ear chondrocytes proliferated faster. Cell proliferation could be further stimulated by the use of serum-free medium with Fibroblast Growth Factor 2 (FGF2) in stead of medium with 10% serum. To evaluate chondrogenic capacity, multiplied chondrocytes were suspended in alginate and implanted subcutaneously in athymic mice. After 8 weeks the constructs demonstrated a proteoglycan-rich matrix that contained collagen type II. Constructs of ear chondrocytes showed a faint staining for elastin. Quantitative RT-PCR revealed that expression of collagen type II was 2-fold upregulated whereas expression of collagen type I was 2-fold down regulated in ear chondrocytes expanded in serum-free medium with FGF2 compared to serum-containing medium. Expression of alkaline phosphatase and collagen type X were low indicating the absence of terminal differentiation. We conclude that ear chondrocytes can be used as donor chondrocytes for cartilage tissue engineering. Furthermore, it may proof to be a promising alternative cell source to engineer cartilage for articular repair.     

Long-term use of polyurethane breast prostheses: a 14-year experience

I have used polyurethane prostheses for the past 14 years, implanting 220 implants into 130 patients who desired breast reconstruction after subcutaneous mastectomy or cancer ablation or simply breast augmentation. I theorize that a polyurethane-covered implant resists contracture, retaining its compressibility because the fibroblasts proliferate into the polyurethane in many different directions. When the fibrils contract, the forces of contracture counterbalance one another, resisting contracture. However, when smooth prostheses are implanted, fibrils are directed in a circular fashion around the implant and naturally contract, leading to firmer breasts. There were 115 prostheses inserted following subcutaneous mastectomy, and 22 percent developed contracted capsules. Seven implants became exposed because of skin necroses; one was removed because of a Staphylococcus infection; and two patients developed a combination of polyurethane and silicone granulomas. These developed only with the earlier implant, where there was shedding of the polyurethane sponge layer and silicone bled from the low-viscosity silicone used in the earlier implants. No granulomas were noted with the currently used Surgitek Replicon implant. Eighty-five breasts were reconstructed after cancer ablation with polyurethane implants, and the contracture rate was 2.3 percent. Other complications were minimal. A smaller group of patients had augmentation mammaplasty, and 20 prostheses were placed in 10 patients. A 15 percent contracture rate was noted in this group. In this study, 82 percent of patients were followed for up to 14 years. Capsular contractures occurred in 30 implants between 1 and 11 years, for an average recurrence at 6.3 years. The overall contracture rate was 13 percent. Other complications were minimal. All implants were placed subcutaneously or subglandularly, and all were drained.     

A 1-year study of osteoinduction in hydroxyapatite-derived biomaterials in an adult sheep model: part I

The study presented here investigated hydroxyapatite biomaterials implanted in soft-tissue sites in adult sheep to determine whether these materials are osteoinductive and whether the rate of osteoinduction can be increased by manipulating the composition and porosity of the implants. For the study, 16.8-mm x 5-mm discs were prepared from mixtures of hydroxyapatite and beta-tricalcium phosphate. Five mixtures of hydroxyapatite-ceramic and hydroxyapatite-cement paste forms were studied: 100 percent hydroxyapatite-ceramic (Interpore), 60 percent hydroxyapatite-ceramic, 100 percent hydroxyapatite-cement paste, 60 percent hydroxyapatite-cement paste, and 20 percent hydroxyapatite-cement paste. Biomaterials were implanted in subcutaneous and intramuscular soft-tissue pockets in 10 adult sheep. Cranial bone grafts of equal dimension were implanted as controls. One year after implantation, the volume of all biomaterials and bone grafts was determined from a computed tomographic scan, and porosity and bone formation were determined using backscatter electron microscopy. Cranial bone and the 20 percent hydroxyapatite-cement paste implants demonstrated significant volume reduction in all sites after 1 year (p < 0.001). No significant difference in volume of the remaining four biomaterials was found. There was no significant change in pore size in the ceramic implants (range, 200 to 300 micro) and in the cement-paste implants containing 60 percent hydroxyapatite or more (range, 3 to 5 nm). Pore size in the cement-paste implants containing 20 percent hydroxyapatite increased significantly with resorption of the tricalcium-phosphate component, reaching a maximum of 200 to 300 micro in the periphery, where the greatest tricalcium-phosphate resorption had occurred. Both ceramic biomaterials demonstrated lamellar bone deposition within well-formed haversian systems through the entire depth of the implants, ranging from a mean of 6.6 percent to 11.7 percent. There was minimal bone formation in the cement-paste implants containing 60 percent hydroxyapatite or more. In contrast, cement-paste implants containing 20 percent hydroxyapatite demonstrated up to 10 percent bone replacement, which was greatest in the periphery of the implants where the greatest tricalcium-phosphate resorption had occurred. This study confirms the occurrence of true osteoinduction within hydroxyapatite-derived biomaterials, when examined using backscatter techniques. In this study, the rate of osteoinduction was greatest when a porous architecture was maintained, which was best achieved in ceramic rather than cement-paste forms of hydroxyapatite. Porosity and resultant bone formation in cement-paste implants can be improved by combining hydroxyapatite with a rapidly resorbing component, such as tricalcium phosphate.