Tissue-engineered Cartilage Implants Research Articles

Curcumin loaded hydrogel with anti-inflammatory activity to promote cartilage regeneration in immunocompetent animals

Stable cartilage regeneration in immunocompetent animals remains a huge challenge, mainly ascribing to the in vivo implantation of tissue-engineered cartilage inevitably arousing inflammatory reactions, resulting in cartilage-specific extracellular matrix erosion, chondrogenic niche destruction, and chondrocyte deterioration. Herein, we developed an anti-inflammatory platform, namely, Cur/GelMA hydrogel, by loading a potent anti-inflammatory drug of curcumin (Cur) into gelatin methacryloyl (GelMA) hydrogel. The Cur/GelMA hydrogel exhibited satisfactory Cur release kinetics in vitro and exerted favorable anti-inflammatory effects when cocultured with lipopolysaccharide-induced RAW264.7 macrophages in vitro. Furthermore, the Cur/GelMA hydrogel showed gratifying biocompatibility and supported cartilage regeneration in vitro when colonized with rabbit- and goat-derived chondrocytes. In addition, the in vitro engineered cartilages in the Cur/GelMA hydrogel were able to maintain a cartilaginous phenotype and achieved stable cartilage regeneration when subcutaneously implanted in autologous rabbits and goats for 2 and 4 weeks compared to the GelMA hydrogel. Furthermore, our data revealed that the in vivo-generated cartilage in the Cur/GelMA group apparently alleviated the inflammatory reaction compared to its GelMA counterpart, suggesting that the locally released Cur endowed the Cur/GelMA hydrogel with potent anti-inflammatory capacity. This study provides a reliable anti-inflammatory platform for stable cartilage regeneration in immunocompetent animals, significantly advancing the clinical application of tissue-engineered cartilage.

Stiffness- and Bioactive Factor-Mediated Protection of Self-Assembled Cartilage against Macrophage Challenge in a Novel Co-Culture System.

Objective:Tissue-engineered cartilage implants must withstand the potential inflammatory and joint loading environment for successful long-term repair of defects. The work’s objectives were to develop a novel, direct cartilage-macrophage co-culture system and to characterize interactions between self-assembled neocartilage and differentially stimulated macrophages.Design:In study 1, it was hypothesized that the proinflammatory response of macrophages would intensify with increasing construct stiffness; it was expected that the neocartilage would display a decrease in mechanical properties after co-culture. In study 2, it was hypothesized that bioactive factors would protect neocartilage properties during macrophage co-culture. Also, it was hypothesized that interleukin 10 (IL-10)-stimulated macrophages would improve neocartilage mechanical properties compared to lipopolysaccharide (LPS)-stimulated macrophages.Results:As hypothesized, stiffer neocartilage elicited a heightened proinflammatory macrophage response, increasing tumor necrosis factor alpha (TNF-α) secretion by 5.47 times when LPS-stimulated compared to construct-only controls. Interestingly, this response did not adversely affect construct properties for the stiffest neocartilage but did correspond to a significant decrease in aggregate modulus for soft and medium stiffness constructs. In addition, bioactive factor-treated constructs were protected from macrophage challenge compared to chondrogenic medium-treated constructs, but IL-10 did not improve neocartilage properties, although stiff constructs appeared to bolster the anti-inflammatory nature of IL-10-stimulated macrophages. However, co-culture of bioactive factor-treated constructs with LPS-treated macrophages reduced TNF-α secretion by over 4 times compared to macrophage-only controls.Conclusions:In conclusion, neocartilage stiffness can mediate macrophage behavior, but stiffness and bioactive factors prevent macrophage-induced degradation. Ultimately, this co-culture system could be utilized for additional studies to develop the burgeoning field of cartilage mechano-immunology.

Accurate Measurements of the Skin Surface Area of the Healthy Auricle and Skin Deficiency in Microtia Patients.

Background:The limited cranial skin covering auricular implants is an important yet underrated factor in auricular reconstruction for both reconstruction surgery and tissue engineering strategies. We report exact measurements on skin deficiency in microtia patients and propose an accessible preoperative method for these measurements.Methods:Plaster ear models (n = 11; male:female = 2:1) of lobular-type microtia patients admitted to the University Medical Center Utrecht in The Netherlands were scanned using a micro-computed tomographic scanner or a cone-beam computed tomographic scanner. The resulting images were converted into mesh models from which the surface area could be calculated.Results:The mean total skin area of an adult-size healthy ear was 47.3 cm2, with 49.0 cm2 in men and 44.3 cm2 in women. Microtia ears averaged 14.5 cm2, with 15.6 cm2 in men and 12.6 cm2 in women. The amount of skin deficiency was 25.4 cm2, with 26.7 cm2 in men and 23.1 cm2 in women.Conclusions:This study proposes a novel method to provide quantitative data on the skin surface area of the healthy adult auricle and the amount of skin deficiency in microtia patients. We demonstrate that the microtia ear has less than 50% of skin available compared with healthy ears. Limited skin availability in microtia patients can lead to healing problems after auricular reconstruction and poses a significant challenge in the development of tissue-engineered cartilage implants. The results of this study could be used to evaluate outcomes and investigate new techniques with regard to tissue-engineered auricular constructs.

The Composition of Engineered Cartilage at the Time of Implantation Determines the Likelihood of Regenerating Tissue with a Normal Collagen Architecture

The biomechanical functionality of articular cartilage is derived from both its biochemical composition and the architecture of the collagen network. Failure to replicate this normal Benninghoff architecture in regenerating articular cartilage may in turn predispose the tissue to failure. In this article, the influence of the maturity (or functionality) of a tissue-engineered construct at the time of implantation into a tibial chondral defect on the likelihood of recapitulating a normal Benninghoff architecture was investigated using a computational model featuring a collagen remodeling algorithm. Such a normal tissue architecture was predicted to form in the intact tibial plateau due to the interplay between the depth-dependent extracellular matrix properties, foremost swelling pressures, and external mechanical loading. In the presence of even small empty defects in the articular surface, the collagen architecture in the surrounding cartilage was predicted to deviate significantly from the native state, indicating a possible predisposition for osteoarthritic changes. These negative alterations were alleviated by the implantation of tissue-engineered cartilage, where a mature implant was predicted to result in the formation of a more native-like collagen architecture than immature implants. The results of this study highlight the importance of cartilage graft functionality to maintain and/or re-establish joint function and suggest that engineering a tissue with a native depth-dependent composition may facilitate the establishment of a normal Benninghoff collagen architecture after implantation into load-bearing defects.

Evaluation of magnetic resonance imaging and clinical outcome after tissue-engineered cartilage implantation: prospective 6-year follow-up study

BackgroundAutologous chondrocyte implantation (ACI) is an important procedure when repairing cartilage defects of the knee. We previously reported several basic studies on tissue-engineered cartilage, and conducted a multicenter clinical study in 2009. In this clinical study, we evaluated the patients' clinical scores and MRI findings before and after tissue-engineered cartilage implantation, and compared the data obtained at 1year and approximately 6years post-implantation. MethodsFourteen patients who underwent implantation of tissue-engineered cartilage to repair cartilage defects of the knee were evaluated. Tissue-engineered cartilage was produced by culturing autologous chondrocytes three dimensionally in atelocollagen gel. The patients were evaluated clinically using the Lysholm score, and the original knee-function score at pre-implantation and at 1year and approximately 6years post-implantation. MRI scans were obtained at the same observation periods. A modified magnetic resonance observation of cartilage repair tissue (MOCART) system was used to quantify clinical efficacy based on the MRI findings. ResultsIn approximately 6years of follow-up, none of the 14 patients reported any subjective symptoms of concern. The mean Lysholm score and the original knee-function score (63.0 ± 10.1, 59.9 ± 5.7) significantly improved at 1year after implantation (86.4 ± 11.8, 94.1 ± 9.2), and were maintained until 6years after implantation (89.8 ± 6.2, 89.9 ± 11.2), although some patients showed deterioration of Lysholm and original knee scores between 1year post-implantation and the final follow-up. The mean MOCART score was 13.2 ± 12.0 pre-implantation, and 62.5 ± 24.7 at 1year and 70.7 ± 22.7 at approximately 6years post-implantation. The MOCART scores at 1year and 6years were significantly higher than the pre-implantation score, but there was no significant difference between the scores at 1 and 6years, indicating that the MRI results at 1year after implantation were maintained for the next 5years. ConclusionsThe clinical scores and MRI findings after implantation of tissue-engineered cartilage were improved at 1year after implantation and were maintained until 6years after implantation.

Human telomerase reverse transcriptase and glucose-regulated protein 78 increase the life span of articular chondrocytes and their repair potential

BackgroundLike all mammalian cells, normal adult chondrocytes have a limited replicative life span, which decreases with age. To facilitate the therapeutic use of chondrocytes from older donors, a method is needed to prolong their life span.MethodsWe transfected chondrocytes with hTERT or GRP78 and cultured them in a 3-dimensional atelocollagen honeycomb-shaped scaffold with a membrane seal. Then, we measured the amount of nuclear DNA and glycosaminoglycans (GAGs) and the expression level of type II collagen as markers of cell proliferation and extracellular matrix formation, respectively, in these cultures. In addition, we allografted this tissue-engineered cartilage into osteochondral defects in old rabbits to assess their repair activity in vivo.ResultsOur results showed different degrees of differentiation in terms of GAG content between chondrocytes from old and young rabbits. Chondrocytes that were cotransfected with hTERT and GRP78 showed higher cellular proliferation and expression of type II collagen than those of nontransfected chondrocytes, regardless of the age of the cartilage donor. In addition, the in vitro growth rates of hTERT- or GRP78-transfected chondrocytes were higher than those of nontransfected chondrocytes, regardless of donor age. In vivo, the tissue-engineered cartilage implants exhibited strong repairing activity, maintained a chondrocyte-specific phenotype, and produced extracellular matrix components.ConclusionsFocal gene delivery to aged articular chondrocytes exhibited strong repairing activity and may be therapeutically useful for articular cartilage regeneration.

Scaffold‐free tissue‐engineered cartilage implants for laryngotracheal reconstruction

Donor site morbidity, including pneumothorax, can be a considerable problem when harvesting cartilage grafts for laryngotracheal reconstruction (LTR). Tissue engineered cartilage may offer a solution to this problem. This study investigated the feasibility of using autologous chondrocytes to tissue-engineer scaffold-free cartilage grafts for LTR in rabbits to avoid degradation that often arises from an inflammatory reaction to scaffold carrier matrix. Animal study. Auricular cartilage was harvested from seven New Zealand white rabbits, the chondrocytes expanded and loaded onto a custom-made bioreactor for 7 to 8 weeks to fabricate autologous scaffold-free cartilage sheets. The sheets were cut to size and used for LTR, and the rabbits were sacrificed 4, 8, and 12 weeks after the LTR and prepared for histology. None of the seven rabbits showed signs of respiratory distress. A smooth, noninflammatory scar was visible intraluminally; the remainder of the tracheal lumen was unremarkable. Histologically, the grafts showed no signs of degradation or inflammatory reaction, were covered with mucosal epithelium, but did show signs of mechanical failure at the implantation site. These results show that autologous chondrocytes can be used to fabricate an implantable sheet of cartilage that retains a cartilage phenotype, becomes integrated, and does not produce a significant inflammatory reaction. These findings suggest that with the design of stronger implants, these implants can be successfully used as a graft for LTR.

Challenging for cartilage repair

Editorial When we look back on the past 15-year history of cartilage repair, it is clear that remarkable progress has been made in this area. There is no doubt that a lot of studies have been carried out on cartilage repair and chondrocytes since Brittebergs' report on ACI in 1994 [1]. I would like to introduce our multi-pronged approach to cartilage repair. After the 1994 report [1], we performed implantation of tissue-engineered cartilage made ex vivo for the treatment of osteochondral defects of the joints, to avoid the leakage of grafted cultured chondrocytes in suspension [2,3]. Sixty knees of 57 patients with full-thickness cartilage defects were followed-up over 5 years. The clinical rating improved significantly after implantation of tissue-engineered cartilage and was maintained for an average of 8.3 years. The arthroscopic findings 2 years after implantation were graded as normal or nearly normal according to the ICRS scale in more than 90% of patients. Biomechanically, stiffness of the graft almost equaled the surrounding normal cartilage (87.9 ~102.5%) 2 years after implantation.

Osteonecrosis of the Knee Treated with a Tissue-Engineered Cartilage and Bone Implant

It is well known that high-dose corticosteroid administration can cause osteonecrosis1,2. Compared with spontaneous osteonecrosis of the knee3, corticosteroid-induced osteonecrosis of the knee tends to be more extensive and often occurs in younger patients. Recently, more attention has been given to regenerative tissue-engineering techniques that make use of autologous cells combined with implants to restore lost or dysfunctional tissues or organs4,5. We have had successful short-term results with use of tissue-engineered cartilage in the treatment of osteochondral defects of the knee6,7. Tissue-engineering techniques, such as the implantation of calcium hydroxyapatite ceramic together with mesenchymal stem cells8-12, have also been applied to bone regeneration. Recently, a new type of hydroxyapatite with interconnected pores (IP-CHA) was introduced for use as a scaffold for bone regeneration13. We present the case of a patient in whom steroid-induced osteonecrosis of the distal part of the femur was treated by implantation of tissue-engineered cartilage together with IP-CHA hybridized with cultured bone-marrow cells. The patient was informed that data concerning the case would be submitted for publication, and she consented. A thirty-six-year-old female office worker had received corticosteroid therapy for the treatment of a connective-tissue disease since 1988. Oral prednisolone had been administered at an initial dosage of 60 mg per day and then gradually reduced. In 1991, the patient began to feel piercing pain in her left knee while walking. In 1993, osteonecrosis of the medial and lateral femoral condyles of the left knee was diagnosed. The knee pain gradually increased despite several nonoperative treatments, and in January 2004 she presented with severe pain in the left knee. In addition, she reported having a clicking sensation in the same knee, which was especially noticeable while walking. …

Mechanical testing of fixation techniques for scaffold‐based tissue‐engineered grafts

Full-thickness defects in articular cartilage can be functionally restored by autologous chondrocyte implantation (ACI). In past years, numerous types of scaffolds for tissue-engineered cartilage implants have been developed and thoroughly characterized. However, the fixation stability of the implants has been rarely investigated despite its well-known importance for successful therapy. In this study, we have mechanically tested the fixation stability of four commonly used biomaterials for ACI attached by four different fixation techniques (unfixed, fibrin glue, chondral suture, and transosseous suture) in situ. Scaffolds based on polyglycolic acid (PGA) and polyglycolic acid and poly-L-lactic acid (PGLA), collagen membranes, and a gel-like matrix material were fixed within rectangular full-thickness cartilage defects of 10 x 15 mm(2) and loaded in tension until failure. Fibrin glue fixation of PGLA-scaffolds withstood a load of 2.18 6 +/- 0.47 N, chondral sutured PGA-scaffolds of 26.29 6 +/- 1.55 N, and transosseous fixed PGA-scaffolds of 38.18 6 +/- 9.53 N. The PGA-scaffold could be loaded highest until failure for all fixation techniques compared to the PGLA-scaffold and collagen membrane. Our findings serve as basis for selecting the most suitable fixation technique for scaffold-based tissue-engineered grafts according to the expected in vivo loads.

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.

Anterior and Posterior Cartilage Graft Dimensions in Successful Laryngotracheal Reconstruction

To describe the dimensions of cartilage grafts used for successful laryngotracheal reconstruction, with the goal of establishing appropriate sizes for "off-the-shelf" tissue-engineered cartilage grafts. A retrospective review of prospectively maintained operative illustrations of a single surgeon's experience. Two tertiary children's hospitals. A consecutive sample of 54 patients (tracheotomized or intubated) with a diagnosis of subglottic stenosis. Each patient underwent anterior (n = 30), posterior (n = 3), or anterior and posterior (n = 22) laryngotracheal reconstruction. Rib cartilage was used in 51 patients and thyroid cartilage was used in 3 patients. Successful or failed extubation. Of the 54 patients, 48 (89%) were successfully decannulated. The mean +/- SEM length of the anterior graft was 20.7 +/- 10.3 mm, and the mean width of the anterior graft was 7.7 +/- 2.5 mm. The mean length of the posterior graft was 13.9 +/- 2.9 mm, and the mean width of the posterior graft was 4.2 +/- 0.9 mm. With the prospect of tissue-engineered cartilage implants becoming available for laryngotracheal reconstruction, the most appropriate templates for designing these implants should be based on the geometric dimensions of grafts carved from native tissues in cases that have been successfully decannulated. Based on our analysis, the use of 2-mm increments for the posterior grafts suggests a set of molds that are 2, 4, and 6 mm wide and 22 mm long. Using 2 x 2-mm increments for the anterior grafts indicates that 36 mold sizes will be sufficient for 90% of predicted cases.

Linkage of chondroitin-sulfate to type I collagen scaffolds stimulates the bioactivity of seeded chondrocytes in vitro

An increasing amount of interest is focused on the potential use of tissue-engineered articular cartilage implants, for repair of defects in the joint surface. In this perspective, various biodegradable scaffolds have been evaluated as a vehicle to deliver chondrocytes into a cartilage defect. This cell–matrix implant should eventually promote regeneration of the traumatized articular joint surface with hyaline cartilage. Successful regeneration can only be achieved with such a tissue-engineered cartilage implant if the seeded cells reveal an appropriate proliferation rate in the biodegradable scaffold together with the production of a new cartilage-specific extracellular matrix. These metabolic parameters can be influenced by the biochemical composition of a cell-delivery scaffold. Further elucidation of specific cell–matrix interactions is important to define the optimal biochemical composition of a cell-delivery vehicle for cartilage repair. In this in vitro study, we investigated the effect of the presence of cartilage-specific glycosaminoglycans in a type I collagen scaffold on the metabolic activity of seeded chondrocytes. Isolated bovine chondrocytes were cultured in porous type I collagen matrices in the presence and absence of covalently attached chondroitin sulfate (CS) up to 14 days. CS did indeed influence the bioactivity of the seeded chondrocytes. Cell proliferation and the total amount of proteoglycans retained in the matrix, were significantly higher ( p<0.001) in type I collagen scaffolds with CS. Light microscopy showed the formation of a more dense cartilaginous layer at the matrix periphery. Scanning electron microscopy revealed an almost complete surfacing of the initially porous surface of both matrices. Histology and reverse transcriptase PCR for various proteoglycan subtypes suggested a good preservation of the chondrocytic phenotype of the seeded cells during culture. The stimulatory potential of CS on both the cell-proliferation and matrix retention, turns this GAG into an interesting biochemical component of a cell-delivery scaffold for use in tissue-engineering articular cartilage.