Cartilage Tissue Engineering

Biodegradable nanofibrous scaffolds serving as an extracellular matrix substitute have been shown to be applicable for cartilage tissue engineering. However, a key challenge in using nanofibrous scaffolds for tissue engineering is that the small pore size limits the infiltration of cells, which may result in uneven cell distribution throughout the scaffold. This study describes an effective method of chondrocyte loading into nanofibrous scaffolds, which combines cell seeding, mixing, and centrifugation to form homogeneous, packed cell-nanofiber composites (CNCs). When the effects of different growth factors are compared, CNCs cultured in medium containing a combination of insulin-like growth factor-I and transforming growth factor-β1 express the highest mRNA levels of collagen type II and aggrecan. Radiolabeling analyses confirm the effect on collagen and sulfated-glycosaminoglycans (sGAG) production. Histology reveals chondrocytes with typical morphology embedded in lacuna-like space throughout the entire structure of the CNC. Upon culturing using a rotary wall vessel bioreactor, CNCs develop into a smooth, glossy cartilage-like tissue, compared to a rough-surface tissue when maintained in a static environment. Bioreactorgrown cartilage constructs produce more total collagen and sGAG, resulting in greater gain in net tissue weight, as well as express cartilage-associated genes, including collagen types II and IX, cartilage oligomeric matrix protein, and aggrecan. In addition, dynamic culture enhances the mechanical property of the engineered cartilage. Taken together, these results indicate the applicability of nanofibrous scaffolds, combined with efficient cell loading and bioreactor technology, for cell-based cartilage tissue engineering.

Background: The developmental history of the chondrocyte results in a cell whose biosynthetic activities are optimized to maintain the concentration and organization of a mechanically functional cartilaginous extracellular matrix. While useful for cartilage tissue engineering studies, the limited supply of healthy autologous chondrocytes may preclude their clinical use. Consequently, multipotential mesenchymal stem cells (MSCs) have been proposed as an alternative cell source. Objective: While MSCs undergo chondrogenesis, few studies have assessed the mechanical integrity of their forming matrix. Furthermore, efficiency of matrix formation must be determined in comparison to healthy chondrocytes from the same donor. Given the scarcity of healthy human tissue, this study determined the feasibility of isolating bovine chondrocytes and MSCs, and examined their long-term maturation in three-dimensional agarose culture. Experimental design: Bovine MSCs were seeded in agarose and induced to undergo chondrogenesis. Mechanical and biochemical properties of MSC-laden constructs were monitored over a 10-week period and compared to those of chondrocytes derived from the same group of animals maintained similarly. Results: Our results show that while chondrogenesis does occur in MSC-laden hydrogels, the amount of the forming matrix and measures of its mechanical properties are lower than that produced by chondrocytes under the same conditions. Furthermore, some important properties, particularly glycosaminoglycan content and equilibrium modulus, plateau with time in MSC-laden constructs, suggesting that diminished capacity is not the result of delayed differentiation. Conclusions: These findings suggest that while MSCs do generate constructs with substantial cartilaginous properties, further optimization must be done to achieve levels similar to those produced by chondrocytes.

Cartilage and osteochondral defects pose a significant challenge in orthopaedics. Tissue engineering has shown promise as a potential method for the treatment of such defects, however, a long lasting repair strategy has yet to be realised. This study focuses on the development of a layered construct for osteochondral repair, fabricated through a novel 'iterative layering' freeze-drying technique. The process involved repeated steps of layer addition followed by freeze-drying, enabling control over material composition, pore size and substrate stiffness in each region of the construct, while also achieving a seamlessly integrated layer structure. The novel construct developed mimics the inherent gradient structure of healthy osteochondral tissue; containing a bone layer composed of type I collagen and hydroxyapatite, an intermediate layer composed of type I collagen, type II collagen and hydroxyapatite, and a cartilaginous region composed of type I collagen, type II collagen and hyaluronic acid. The material properties were designed to provide the biological cues required to encourage infiltration of host cells from the bone marrow while the biomechanical properties were designed to provide an environment optimised to promote differentiation of these cells towards the required lineage in each region. This novel osteochondral graft was shown to have a seamlessly integrated layer structure, high levels of porosity (>97%), a homogenous pore structure and a high degree of pore interconnectivity. Moreover, homogenous cellular distribution throughout the entire construct was evident following in vitro culture demonstrating the potential of this multi-layered scaffold as an advanced strategy for osteochondral defect repair.

Background: Synthetic-and naturally derived-biodegradable polymers have been widely used to construct scaffolds for cartilage tissue engineering. Poly(lactic-co-glycolic acid) (PLGA) are bioresorbable and biocompatible, rendering them as a promising tool for clinical application. To minimize cells lost during the seeding procedure, we used the natural polymer fibrin to immobilize cells and to provide homogenous cells distribution in PLGA scaffolds. We evaluated in vitro chondrogenesis of rabbit articular chondrocytes in PLGA scaffolds using fibrin as cell transplantation matrix. Methods: PLGA scaffolds were soaked in chondrocytes-fibrin suspension (1 × 10 6 cells/scaffold) and polymerized by dropping thrombin-calcium chloride (CaCl 2 ) solution. PLGA-seeded chondrocytes was used as control. All constructs were cultured for a maximum of 21 days. Cell proliferation activity was measured at 1, 3, 7, 14 and 21 days in vitro using 3-(4,5-dimethylthiazole-2-yl)-2-, 5-diphenyltetrazolium-bromide (MTT) assay. Morphological observation, histology, immunohistochemistry (IHC), gene expression and sulphated-glycosaminoglycan (sGAG) analyses were performed at each time point of 1, 2 and 3 weeks to elucidate in vitro cartilage development and deposition of cartilage-specific extracellular matrix (ECM). Results: Cell proliferation activity was gradually increased from day-1 until day-14 and declined by day-21. A significant cartilaginous tissue formation was detected as early as 2-week in fibrin/PLGA hybrid construct as confirmed by the presence of cartilage-isolated cells and lacunae embedded within basophilic ECM. Cartilage formation was remarkably evidenced after 3 weeks. Presence of cartilage-specific proteoglycan and glycosaminoglycan (GAG) in fibrin/PLGA hybrid constructs were confirmed by positive Safranin O and Alcian Blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrix. Chondrogenic properties were further demonstrated by the expression of genes encoded for cartilage-specific markers, collagen type II and aggrecan core protein. Interestingly, suppression of cartilage dedifferentiation marker; collagen type I was observed after 2 and 3 weeks of in vitro culture. The sulphatedglycosaminoglycan (sGAG) production in fibrin/PLGA was significantly higher than in PLGA. Fibrin/PLGA promotes early in vitro chondrogenesis of rabbit articular chondrocytes. This study suggests that fibrin/PLGA may serve as a potential cell delivery vehicle and a structural basis for in vitro tissue-engineered articular cartilage.

Background. is study aims to compare the e ects of platelet-rich plasma (PRP) alone or in combination with adipose-derived mesenchymal stem cells (AD-MSCs) in patients a ected by cartilage defects, undergoing knee arthroscopic microfracture. Methods. irty-eight patients diagnosed with a knee monocompartmental cartilage defect (Outerbridge grade IV) on the MRI, underwent an arthroscopic procedure. After the con rmation of the lesion, they all received the same bone marrow stimulation technique (microfracture) and were randomized into two groups: the rst one had additional PRP injection (group A), while the second received PRP and AD-MSC injection (group B). Knee assessment and pain score were documented with Knee Injury Osteoarthritis Outcome Score (KOOS), International Knee Documentation Committee (IKDC) score, Short-Form (SF) 12, and Visual Analogue Scale (VAS) before the treatment and at 1, 3, 6, and 12 months of follow-up postoperatively. An additional arthroscopic procedure, performed in four patients for a subsequent meniscal lesion, let us evaluate cartilage evolution by performing a macro/microscopical assessment on cartilage biopsy specimens. Results. At the 12-month follow-up, both groups showed a comparable functional improvement. e scores on the IKDC form, KOOS, pain VAS, and SF-12 signi cantly improved from baseline (p < 0.05) to 12 months postoperatively in both treatment groups. e four second-look arthroscopies showed a complete repair of the articular defects by smooth solid cartilage layer, with a good chondrocytic population, in both groups. A thick smooth hyaline-like cartilage with a predominantly viable cell population and normal mineralization (a form closely resembling native tissue) was observed in group B. Conclusions Modern regenerative medicine techniques, such as PRP and AD-MSC, associated with traditional arthroscopic bone marrow stimulating techniques, seem to enhance cartilage restoration ability. e preliminary results of this pilot study encourage the synergic use of these regenerative modulating systems to improve the quality of the regenerated cartilage.

To date, no single approach to the treatment of osteochondral defects has resulted in satisfactory long-term outcomes, especially in a young and active human population. Emerging innovative tissue engineering strategies, including the use of composite scaffolds, novel cell sources and bioreactors, have shown promising results. However, these techniques need to be validated in translational animal models before they can be implemented in clinical practice. The aim of the present study was to analyse morphological and microarchitectural parameters during subchondral bone repair following transplantation of bioreactormanufactured autologous osteochondral grafts in a sheep model. Animals were divided into 4 treatment groups: nasal chondrocyte (NC) autologous osteochondral grafts, articular chondrocyte (AC) autologous osteochondral grafts, cell-free scaffolds (CFS) and empty defects (EDs). After 6 weeks, 3 months and 12 months, bone remodelling was assessed by histology and micro-computed tomography (µCT). Although gradual remodelling and subchondral bone repair were seen in all groups across the time points, the best results were observed in the NC group. This was evidenced by the extent of new tissue formation and its best integration into the surrounding tissue in the NC group at all time points. This also suggested that nasal septum chondrocyte-seeded grafts adapted well to the biomechanical conditions of the loaded joint surface.

In cartilaginous tissues, perichondrium cambium layer may be the source of new cartilage. Human nasal septal perichondrium is considered to be a homogeneous structure in which some authors do not recognize the perichondrium internal zone or the cambium layer as a layer distinct from adjacent cartilage surface. In the present study, we isolated a chondrogenic cell population from human nasal septal cartilage surface zone. Nasoseptal chondrogenic cells were positive for surface markers described for mesenchymal stem cells, with exception of CD146, a perivascular cell marker, which is consistent with their avascular niche in cartilage. Although only Sox-9 was constitutively expressed, they also revealed osteogenic and chondrogenic, but not adipogenic, potentials in vitro, suggesting a more restricted lineage potential compared to mesenchymal stem cells. Interestingly, even in absence of chondrogenic growth factors in the pellet culture system, nasoseptal chondrogenic cells had a capacity to synthesize sulfated glycosaminoglycans, large amounts of collagen type II and to a lesser extent collagen type I. The spontaneous chondrogenic potential of this population of cells indicates that they may be a possible source for cartilage tissue engineering. Besides, the pellet culture system using nasoseptal chondrogenic cells may also be a model for studies of chondrogenesis.

Extracellular matrix (ECM) materials have diverse physiological functions by themselves and can also act as reservoirs of cytokines and growth factors, so that they can affect the cell phenotype, attachment, migration and proliferation of cells. In this study, an ECM scaffold derived from porcine cartilage was evaluated for whether it can support and maintain chondrogenesis of rabbit mesenchymal stem cells (rMSCs) in vitro and in the nude mouse model in vivo. The porcine ECM scaffold was compared to a polyglycolic acid (PGA) scaffold and an MSC pellet as a control group. In an in vitro environment until 4 weeks, the ECM scaffold evoked chondrogenic differentiation of rMSCs earlier and produced more cartilaginous tissues than the PGA scaffold. Next, rMSCs in each scaffold were preconditioned with chondrogenic media in vitro for 1 week and implanted in the backs of nude mice for 6 weeks. The initially formed cartilaginous tissues turned into bone matrix with time centripetally from the outside of the region as observed by Safranin-O and von Kossa stains. This phenomenon progressed much more rapidly in the PGA group than in the ECM group. In the ECM group, the chondrogenic phenotypes of rMSCs were also maintained longer than in the PGA group. The loss of chondrogenic phenotypes was accompanied by the calcification of matrix, and hypertrophic changes by immunohistochemistry for osteocalcin and collagen type I and X. Blood vessel invasion took place more deeply and intensively in the PGA group. These results suggest that the ECM scaffold not only strongly supports chondrogenic differentiation of rMSCs, but also helps maintain its phenotype in vivo. We speculate that the ECM scaffold provides rMSCs with a favorable, native cartilage-like environment and therefore can be a promising tool for cartilage tissue engineering.

Adult stem cells (ASC) have been found in many tissues and are of great therapeutic potential due to their capability of differentiation. However, ASC comprise only a small fraction of the tissues. In order to use ASC for therapeutic purposes, it is important to obtain relatively pure stem cells in large quantities. Current methods for stem cell purification are mainly based on marker-dependent cell sorting techniques, which have various technical difficulties. In this study, we have attempted to develop novel conditions to favor the growth of the dental follicle stem cells (DFSC) such that the resultant cell populations are enriched in stem cells. Specifically, a heterogeneous dental follicle cell (H-DFC) population containing stem cells and homogenous non-stem cell dental follicle cell population were cultured at 1% or 5% hypoxic conditions. Only the heterogeneous population could increase proliferation in the hypoxic condition whereas the homogenous DFC did not change their proli...

Introduction: Osteoarthritis is a degenerative disease that has many public health problems. Interleukin 10 (IL-10) and tumor necrosis factor alfa (TNF-α) are considered as the main factors regulating inflammation and pathology of knee osteoarthritis. This study was done to investigate the effects of mesenchymal stem cells (MSCs) and aerobic training on inflammatory biomarkers (IL-10 and TNF-α) in the brain tissue of osteoarthritic rats. Materials and Methods: In this experimental study, 48 male Wistar rats were randomly divided into six groups (n=8 in each group) as follows: 1. Naive (healthy control), 2. Osteoarthritis (Ost), 3. Osteoarthritis + saline (Sal), 4. Osteoarthritis + mesenchymal cells (Mes), 5. Osteoarthritis + training (Tra), and 6. Osteoarthritis + training + mesenchymal cells (Tra + Mes). An essential aerobic exercise program was performed for eight weeks as follows: Speed: 15-22 meters per minute; Slope: 0 o for 25-64 minutes. Inflammatory biomarkers, including TNF-α and IL-10 cytokines, in the brain tissue of rats, were measured using enzyme-linked immunosorbent assay. Results: The combination of training and treatment with MSCs in the brain of osteoarthritic rats significantly increased the level of IL-10 in comparison with the use of MSCs or aerobic exercise alone. Moreover, the combined use of exercise and MSCs caused a significant decrease in TNF-α concentration. Conclusion: It seems that the combined use of MSCs and eight weeks of aerobic exercise improves the concentration of inflammatory biomarkers in the brain of a rat model of osteoarthritis.

Objective: The purpose of this review is to gain insight into the latest methods of articular cartilage implantation (ACI) and to detail where they are in the Food and Drug Administration approval and regulatory process. Design: A PubMed search was performed using the phrase "Autologous Chondrocyte Implantation" alone and with the words second generation and third generation. Additionally, clinicaltrials.gov was searched for the names of the seven specific procedures and the parent company websites were referenced. Results: Two-Stage Techniques: BioCart II uses a FGF2v1 culture and a fibrinogen, thrombin matrix, whereas Hyalograft-C uses a Hyaff 11 matrix. MACI uses a collagen I/III matrix. Cartipatch consists of an agarose-alginate hydrogel. Neocart uses a high-pressure bioreactor for culturing with a type I collagen matrix. ChondroCelect makes use of a gene expression analysis to predict chondrocyte proliferation and has demonstrated significant clinical improvement, but failed to show superiority to microfracture in a phase III trial. One Step Technique: CAIS is an ACI procedure where harvested cartilage is minced and implanted into a matrix for defect filling. Conclusion: As full thickness defects in articular cartilage continue to pose a challenge to treat, new methods of repair are being researched. Later generation ACI has been developed to address the prevalence of fibrocartilage with microfracture and the complications associated with the periosteal flap of first generation ACI such as periosteal hypertrophy. The procedures and products reviewed here represent advances in tissue engineering, scaffolds and autologous chondrocyte culturing that may hold promise in our quest to alter the natural history of symptomatic chondral disease.

The purpose of this review is to gain insight into the latest methods of articular cartilage implantation (ACI) and to detail where they are in the Food and Drug Administration approval and regulatory process. A PubMed search was performed using the phrase &quot;Autologous Chondrocyte Implantation&quot; alone and with the words second generation and third generation. Additionally, clinicaltrials.gov was searched for the names of the seven specific procedures and the parent company websites were referenced. Two-Stage Techniques: BioCart II uses a FGF2v1 culture and a fibrinogen, thrombin matrix, whereas Hyalograft-C uses a Hyaff 11 matrix. MACI uses a collagen I/III matrix. Cartipatch consists of an agarose-alginate hydrogel. Neocart uses a high-pressure bioreactor for culturing with a type I collagen matrix. ChondroCelect makes use of a gene expression analysis to predict chondrocyte proliferation and has demonstrated significant clinical improvement, but failed to show superiority ...

Gellan gum is a polysaccharide that we have previously proposed for applications in the cartilage tissue engineering field. In this work, gellan gum hydrogels were tested for their ability to be used as injectable systems using simple processing methods, able to deliver and maintain chondrocytes by in situ gelation, and support cell viability and production of extracellular matrix (ECM). Rheological measurements determined that the sol-gel transition occurred near the body temperature at 398C, upon temperature decrease, in approximately 20 s. Gellan gum discs shows a storage compression modulus of around 80 kPa at a frequency of 1 Hz by dynamic mechanical analysis. Human articular chondrocytes were encapsulated in the gels, cultured in vitro for total periods of 56 days, and analyzed for cell viability and ECM production. Calcein AM staining showed that cell kept viable after 14 days and the histological analysis and real-time quantitative polymerase chain reaction revealed that hyaline-like cartilage ECM was synthesized. Finally, the in vivo performance of the gellan gum hydrogels, in terms of induced inflammatory reaction and integration into the host tissue, was evaluated by subcutaneous implantation in Balb=c mice for 21 days. Histological analysis showed a residual fibrotic capsule at the end of the experiments. Dynamic mechanical analysis revealed that the gels were stable throughout the experiments while evidencing a tendency for decreasing mechanical properties, which was consistent with weight measurements. Altogether, the results demonstrate the adequacy of gellan gum hydrogels processed by simple methods for noninvasive injectable applications toward the formation of a functional cartilage tissue-engineered construct and originally report the preliminary response of a living organism to the subcutaneous implantation of the gellan gum hydrogels. These are the two novel features of this work.

Tissue engineering may provide a technique to generate cartilage grafts for laryngotracheal reconstruction in children. The present study used a rabbit model to characterize cartilage generated by a candidate tissue engineering approach to determine, under baseline conditions, which chondrocytes in the rabbit produce tissue-engineered cartilage suitable for in vivo testing in laryngotracheal reconstruction. We characterized tissue-engineered cartilage generated in perfused bioreactor chambers from three sources of rabbit chondrocytes: articular, auricular, and nasal cartilage. Biomechanical testing and histological, immunohistochemical, and biochemical assays were performed to determine equilibrium unconfined compression (Young's) modulus, and biochemical composition and structure. We found that cartilage samples generated from articular or nasal chondrocytes lacked the mechanical integrity and stiffness necessary for completion of the biomechanical testing, but five of six auricular samples completed the biomechanical testing (moduli of 210 ± 93 kPa in two samples at 3 weeks and 100 ± 65 kPa in three samples at 6 weeks). Auricular samples showed more consistent staining for proteoglycans and collagen II and had significantly higher glycosaminoglycan (GAG) content and concentration and higher collagen content than articular or nasal samples. In addition, the delayed gadolinium enhanced MRI of cartilage (dGEMRIC) method revealed variations in GAG spatial distribution in auricular samples that were not present in articular or nasal samples. The results indicate that, for the candidate tissue engineering approach under baseline conditions, only rabbit auricular chondrocytes produce tissue-engineered cartilage suitable for in vivotesting in laryngotracheal reconstruction. The results also suggest that this and similar tissue engineering approaches must be optimized for each potential source of chondrocytes.