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Human mesenchymal stem cell (hMSC)-based chondrogenesis is a key process used to develop tissue engineered cartilage constructs from stem cells, but the resulting constructs have inferior biochemical and biomechanical properties compared to native articular cartilage. Transforming growth factor β containing medium is commonly applied to cell layers of hMSCs, which aggregate upon centrifugation to form 3-D constructs. The aggregation process leads to a high cell density condition, which can cause nutrient limitations during long-term culture and, subsequently, inferior quality of tissue engineered constructs. Our objective is to modulate the aggregation process by targeting RhoA/ROCK signaling pathway, the chief modulator of actomyosin contractility, to enhance the end quality of the engineered constructs. Through ROCK inhibition, repression of cytoskeletal tension in chondrogenic hMSCs was achieved along with less dense aggregates with enhanced transport properties. ROCK inhibition also led to significantly increased cartilaginous extracellular matrix accumulation. These findings can be used to create an improved microenvironment for hMSC-derived tissue engineered cartilage culture. We expect that these findings will ultimately lead to improved cartilaginous tissue development from hMSCs.

Novel strategies are urgently required to facilitate regeneration of entire bones lost due to trauma or disease. In this study, we present a novel framework for the regeneration of whole bones by tissue engineering anatomically shaped hypertrophic cartilaginous grafts in vitro that subsequently drive endochondral bone formation in vivo. To realize this, we first fabricated molds from digitized images to generate mesenchymal stem cell-laden alginate hydrogels in the shape of different bones (the temporomandibular joint [TMJ] condyle and the distal phalanx). These constructs could be stimulated in vitro to generate anatomically shaped hypertrophic cartilaginous tissues that had begun to calcify around their periphery. Constructs were then formed into the shape of the distal phalanx to create the hypertrophic precursor of the osseous component of an engineered long bone. A layer of cartilage engineered through self-assembly of chondrocytes served as the articular surface of these constructs. Following chondrogenic priming and subcutaneous implantation, the hypertrophic phase of the engineered phalanx underwent endochondral ossification, leading to the generation of a vascularized bone integrated with a covering layer of stable articular cartilage. Furthermore, spatial bone deposition within the construct could be modulated by altering the architecture of the osseous component before implantation. These findings open up new horizons to whole limb regeneration by recapitulating key aspects of normal bone development.

Adipose-derived stem cells (ASCs) have captured growing interests for cartilage regeneration. Although ASCs chondrogenesis can be stimulated by genetic modification, whether genetically engineered ASCs hold promise for the cartilaginous tissue formation remains to be explored. Since baculovirus (an emerging gene delivery vector) effectively transduced ASCs and transforming growth factor β3 (TGF-β3) was recently shown to induce ASCs chondrogenesis more potently than TGF-β1, we constructed a baculoviral vector (Bac-CT3W) to encode TGF-β3. The Bac-CT3W-transduced ASCs expressed TGF-β3 robustly and substantiated the chondrogenesis of ASCs cultured in monolayer and in porous scaffolds. Culture of the transduced cell/scaffold constructs in the rotating-shaft bioreactor (RSB) under hypoxic and perfusion conditions for 2 weeks further augmented the ASCs chondrogenesis and deposition of cartilage-specific collagen II and glycosaminoglycans, leading to the formation of cartilage-like tissues with hyaline appearance and compressive modulus approaching 62% of the native articular cartilage. Intriguingly, prolonged culture to 3 or 4 weeks failed to further augment the construct growth, probably due to the scaffold degradation. Altogether, baculovirus-mediated TGF-β3 expression in ASCs in conjunction with dynamic culture in the RSB for 2 weeks synergistically ameliorated the ASCs chondrogenesis and formation of cartilaginous tissues, representing a novel approach to producing engineered cartilages.

In cartilage tissue engineering an optimized culture system, maintaining an appropriate extracellular environment (e.g., pH of media), can increase cell proliferation and extracellular matrix (ECM) accumulation. We have previously reported on a continuous-flow bioreactor that improves tissue growth by supplying the cells with a near infinite supply of medium. Previous studies have observed that acidic environments reduce ECM synthesis and chondrocyte proliferation. Hence, in this study we investigated the combined effects of a continuous culture system (bioreactor) together with additional buffering agents (e.g., sodium bicarbonate [NaHCO₃]) on cartilaginous tissue growth in vitro. Isolated bovine chondrocytes were grown in three-dimensional cultures, either in static conditions or in a continuous-flow bioreactor, in media with or without NaHCO₃. Tissue constructs cultivated in the bioreactor with NaHCO₃-supplemented media were characterized with significantly increased (p0.05) ECM accumulation (glycosaminoglycans a 98-fold increase; collagen a 25-fold increase) and a 13-fold increase in cell proliferation, in comparison with static cultures. Additionally, constructs grown in the bioreactor with NaHCO₃-supplemented media were significantly thicker than all other constructs (p0.05). Further, the chondrocytes from the primary construct expanded and synthesized ECM, forming a secondary construct without a separate expansion phase, with a diameter and thickness of 4 mm and 0.72 mm respectively. Tissue outgrowth was negligible in all other culturing conditions. Thus this study demonstrates the advantage of employing a continuous flow bioreactor coupled with NaHCO₃ supplemented media for articular cartilage tissue engineering.

Tissue engineering is a promising approach for articular cartilage repair; however, it is challenging to produce adequate amounts of tissue in vitro from the limited number of cells that can be extracted from an individual. Relatively few cell expansion methods exist without the problems of de-differentiation and/or loss of potency. Recently, however, several studies have noted the benefits of three-dimensional (3D) over monolayer expansion, but the ability of 3D expanded chondrocytes to synthesize cartilaginous tissue constructs has not been demonstrated. Thus, the purpose of this study was to compare the properties of engineered cartilage constructs from expanded cells (monolayer and 3D microcarriers) to those developed from primary chondrocytes. Isolated bovine chondrocytes were grown for 3 weeks in either monolayer (T-Flasks) or 3D microcarrier (Cytodex 3) expansion culture. Expanded and isolated primary cells were then seeded in high density culture on Millicell™ filters for 4 weeks to evaluate the ability to synthesize cartilaginous tissue. While microcarrier expansion was twice as effective as monolayer expansion (microcarrier: 110-fold increase, monolayer: 52-fold increase), the expanded cells (monolayer and 3D microcarrier) were not effectively able to synthesize cartilaginous tissue in vitro. Tissues developed from primary cells were substantially thicker and accumulated significantly more extracellular matrix (proteoglycan content: 156%-292% increase; collagen content: 70%-191% increase). These results were attributed to phenotypic changes experienced during the expansion phase. Monolayer expanded chondrocytes lost their native morphology within 1 week, whereas microcarrier-expanded cells were spreading by 3 weeks of expansion. While the use of 3D microcarriers can lead to large cellular yields, preservation of chondrogenic phenotype during expansion is required in order to synthesize cartilaginous tissue.

The development of porous scaffolds having both high porosity and strong mechanical strength for tissue engineering and regenerative medicine has been quite challenging. A novel hybrid poly(L-lactic acid) (PLLA)-collagen hybrid sponge was developed by enclosing collagen sponge in a cup-shaped PLLA sponge to meet the necessary requirements. Collagen sponge was formed in the center of the PLLA sponge cup, and collagen microsponges were formed in the pores of the PLLA sponge cup. The PLLA-collagen hybrid sponge showed higher mechanical strength than did those of the PLLA sponge cup and collagen sponge. The porosity of the PLLA-collagen hybrid sponge was greater than that of the PLLA sponge cup. The cup-shaped PLLA sponge skeleton provided the hybrid sponge with high mechanical strength and protected against cell leakage during cell seeding, while the central collagen sponge contributed to high porosity, and facilitated cell adhesion and distribution in the hybrid sponge. Cartilaginous tissue was successfully regenerated when chondrocytes were cultured in the hybrid sponge. This method of hybridization will provide a new technique for the preparation of functional porous scaffolds for tissue engineering.

Many cartilaginous tissues such as intervertebral disc (IVD) display a heterogeneous collagen microstructure that results in mechanical anisotropy. These structures are responsible for mechanical function of the tissue and regulate cellular interactions and metabolic responses of cells embedded within these tissues. Using collagen gels seeded with ovine annulus fibrosus cells, constructs of varying structure and heterogeneity were created to mimic the circumferential alignment of the IVD. Alignment was induced within gels by contracting annular gels around an inner boundary using both a polyethylene center and alginate center to create a composite engineered IVD. Collagen alignment and heterogeneity were measured using second harmonic generation microscopy. Decreasing initial collagen density from 2.5 mg/mL to 1 mg/mL produced greater contraction of constructs, resulting in gels that were 55% and 6.2% of the original area after culture, respectively. As a result, more alignment occurred in annular-shaped 1 mg/mL gels compared with 2.5 mg/mL gels (p

Tissue-engineered bone (TEB) has shown to be an effective alternative to conventional gold-standard autogenous bone for the repair of critically sized bone defects (CSBD). Moderate axial interfragmentary movement (IFM) has been shown to promote bone healing in conventional models. This study explored the use of IFM to enhance the capacity of TEB in the repair of CSBD using a goat model. CSBD were created in a goat model. Dynamic intramedullary rods designed to supply axial IFMs within 10% of the interfragmentary strain were used to stabilize CSBD goat femur models, whose bone defects were filled with TEB. Bone regeneration was evaluated using radionuclide bone imaging, roentgenographic analysis, periosteal callus area, computed tomography value score, biomechanical analysis, and histological observation. Compared with the static intramedullary rods, the dynamic intramedullary rod group showed an increase in early-stage callus formation and blood supply to the callus tissue, better differentiation of fibrous and cartilaginous tissue into bone tissue, improved strength and stiffness of callus tissue in late-stage healing, and overall better functional recovery of the goat femur. This showed that moderate axial IFM could promote the osteogenesis and reconstruction of TEB in vivo.

Tissue engineering is a promising approach, not only for cartilage, but also for osteochondral repair. Recent studies have demonstrated that scaffold-free cartilaginous tissue can be engineered using the alginate-recovered-chondrocyte (ARC) method. This method has also been applied to form osteochondral tissue using bovine articular chondrocytes and coralline hydroxyapatite (HA). The purpose of this study was to test whether osteochondral tissue, fabricated in vitro using the ARC method combined with a block of HA, would undergo maturation in vivo using a subcutaneous model in immunodeficient mice. Articular chondrocytes were isolated from the cartilage of New Zealand white rabbits and cultured in alginate beads. The cells with their associated matrix were recovered by dissolving the alginate beads with a sodium citrate buffer, resuspended in media and seeded onto a porous HA block. After 4 weeks of culture, some samples were analyzed, and others were implanted into subcutaneous pockets in nude mice. The analysis involved removing the cartilage portion of the de novo-formed ARC-HA graft and performing biochemical and histological examinations. Some samples were subjected to nondecalcified histology. Histological and immunohistochemical analyses of cartilaginous tissue were performed at 0, 2, 4, and 8 weeks after implantation. Biochemical characteristics were examined at 0, 4, and 8 weeks. The size and shape of the implanted ARC osteochondral tissue changed with time. The histological and immunohistochemical examination of the tissue revealed that it contained a cartilage-like matrix that stained strongly with Toluidine blue and for collagen type II. The proteoglycan (PG) content had increased significantly at 4 weeks from baseline. However, by 8 weeks, the PG content had decreased from 4 weeks. The results presented here represent a possible approach to form a tissue-engineered osteochondral implant. Further studies are needed to improve biomechanical properties of the osteochondral implant to be suitable for surgical transplantation.

Tissue engineering is a promising approach for articular cartilage repair; however, it still has proven a challenge to produce substantial quantities of tissue from the limited number of cells that can be extracted from a single individual. Although several approaches have been investigated to enhance the production of cartilaginous tissue in vitro, relatively few techniques exist to reliably increase the population of cells needed for this approach. Alternatively, a single modulator of chondrocyte function, such as the C-type natriuretic peptide (CNP), may serve to address both of these issues. CNP is expressed in the growth plate and regulates cartilage growth through chondrocyte proliferation and differentiation. Thus, the purpose of this study was to determine the effects of CNP stimulation on tissue-engineered cartilage. Isolated bovine articular chondrocytes were seeded on Millicell filters and cultured in the presence of CNP (10 pM to 10 nM) for 4 weeks. Stimulation with CNP resulted in differential effects depending on the dose of the peptide. Low doses of CNP (10 to 100 pM) elicited chondrocyte proliferation with a maximal response observed at 100 pM (43% increase in cellularity). However, high doses of CNP (10 nM) stimulated matrix deposition (36% and 137% increase in proteoglycans and collagen) without an associated change in tissue cellularity. CNP stimulation also downregulated the expression of type X collagen, an early hypertrophic marker associated with endochondral ossification. Thus, by regulating the dose of CNP, it may be possible to produce engineered tissue from the limited number of cells that can be reasonably extracted from a single individual for therapeutic purposes.

The use of either a hydrogel or a solid polymeric scaffold alone is often associated with distinct drawbacks in many tissue engineering applications. Therefore, in this study, we investigated the potential of a combination of long-term stable fibrin gels and polyurethane scaffolds for cartilage engineering. Primary bovine chondrocytes were suspended in fibrin gel and subsequently injected into a polycaprolactone-based polyurethane scaffold. Cells were homogeneously distributed within this composite system and produced high amounts of cartilage-specific extracellular matrix (ECM) components, namely glycosaminoglycans (GAGs) and collagen type II, within 4 weeks of in vitro culture. In contrast, cells seeded directly onto the scaffold without fibrin resulted in a lower seeding efficiency and distinctly less homogeneous matrix distribution. Cell-fibrin-scaffold constructs implanted into the back of nude mice promoted the formation of adequate engineered cartilaginous tissue within the scaffold after 1, 3, and 6 months in vivo, containing evenly distributed ECM components, such as GAGs and collagen. Again, in constructs seeded without fibrin, histology showed an inhomogeneous and, thus, not adequate ECM distribution compared to seeding with fibrin, even after 6 months in vivo. Strikingly, a precultivation for 1 week in vitro elicited similar results in vivo compared to precultivation for 4 weeks; that is, a precultivation for longer than 1 week did not enhance tissue development. The presented composite system is suggested as a promising alternative toward clinical application of engineered cartilaginous tissue for plastic and reconstructive surgery.

Here we present the development of a visual evaluation system for routine assessment of in vitro-engineered cartilaginous tissue. Neocartilage was produced by culturing human articular chondrocytes in pellet culture systems or in a scaffold-free bioreactor system. All engineered tissues were embedded in paraffin and were sectioned and stained with Safranin O-fast green. The evaluation of each sample was broken into 3 categories (uniformity and intensity of Safranin O stain, distance between cells/amount of matrix produced, and cell morphology), and each category had 4 components with a score ranging from 0 to 3. Three observers evaluated each sample, and the new system was independently tested against an objective computer-based histomorphometry system. Pellets were also assessed biochemically for glycosaminoglycan (GAG) content. Pellet histology scores correlated significantly with GAG contents and were in agreement with the computer-based histomorphometry system. This system allows a valid and rapid assessment of in vitro-generated cartilaginous tissue that has a relevant association with objective parameters indicative of cartilage quality.

Tissue engineering of articular cartilage is a promising alternative for cartilage repair. However, it has been difficult to develop tissue in vitro that mimicks native cartilage. Cartilaginous tissue formed in vitro does not accumulate enough extracellular matrix, is deficient in collagen, and possesses only a fraction of the mechanical properties of native cartilage. In this study, we investigated whether long-term intermittent compressive stimulation would improve the quality of the generated tissue. Chondrocyte cultures were established on the surface of porous calcium polyphosphate substrates and allowed to form cartilaginous tissue. In vitro-formed tissues were subjected to different stimulation protocols for 1 week. The optimal mechanical stimulation parameters identified in this short-term study were then applied to the cultures for up to 4 weeks. Mechanical stimulation applied at a 5% compressive amplitude at a frequency of 1 Hz for 400 cycles every second day resulted in the greatest increase in collagen synthesis (37 +/- 9% over control) while not significantly affecting proteoglycan synthesis (2 +/- 8% over control). This condition, applied to the chondrocyte cultures for 4 weeks, resulted in a significant increase in the amount of tissue that formed (stimulated, 2.4 +/- 0.2 mg dry wt; unstimulated, 1.61 +/- 0.08 mg dry wt). Stimulated tissues contained approximately 40% more collagen (stimulated, 590 +/- 58 microg; unstimulated, 420 +/- 42 microg), and 30% more proteoglycans (stimulated, 393 +/- 34 microg; unstimulated, 302 +/- 32 microg) as well as displaying a 2- to 3-fold increase in compressive mechanical properties (maximal equilibrium stress: stimulated, 10 +/- 1 kPa; unstimulated, 5 +/- 1 kPa; maximal equilibrium modulus: stimulated, 80 +/- 23 kPa; unstimulated, 24 +/- 6 kPa). The results of this study demonstrate that intermittent mechanical stimulation can increase collagen synthesis and, when applied over a 4-week period, can accelerate extracellular matrix accumulation as well as improve the material properties of the developed tissue. Interestingly, only short periods of mechanical stimulation (6 min every second day) were needed to affect the quality of cartilaginous tissue formed in vitro.

Our previous study showed that cartilaginous tissue can be engineered in vitro with articular chondrocytes and poly(N-isopropylacrylamide)-grafted gelatin. This short-term in vivo study for cartilage repair was performed to screen a candidate method for a long-term study. In our previous in vitro study, however, two potential problems with the tissue-engineered cartilage were identified: (1). leakage of the transplant due to temperature decline and (2). concave deformation of transplant due to compressive loading. To solve these problems, we investigated in this study the usefulness of suturing with two different covering materials (periosteum or collagen film) and preculturing an engineered tissue for 2 weeks. PNIPAAm-gelatin-based engineered cartilage samples were evaluated at 5 weeks after operation by gross and microscopic examination. Leakage occurred only in specimens without precultured tissue and with a collagen film. Minimal surface deformation occurred in all specimens with precultured tissue. The score on gross examination showed that transplants with precultured tissue acquired a higher score than did the others. Histological evaluation showed a minimal foreign-body response of PNIPAAm-gelatin in all specimens and higher maturity as a cartilaginous tissue in specimens with precultured tissue. These results indicate that transplantation with precultured tissue may be a suitable method for a long-term in vivo study.

The implantation of laboratory-grown tissue offers a valuable alternative approach to the treatment of cartilage defects. Procuring sufficient cell numbers for such tissue-engineered cartilage is a major problem since amplification of chondrocytes in culture typically leads to loss of normal cell phenotype yielding cartilage of inferior quality. In an effort to overcome this problem, we endeavored to regain the differentiated phenotype of chondrocytes after extensive proliferation in monolayer culture by modulating cell morphology and oxygen tension towards the in vivo state. Passaged cells were encapsulated in alginate hydrogel in an effort to regain the more rounded shape characteristic of differentiated chondrocytes. These cultures were exposed to reduced (5%-i.e., physiological), or control (20%) oxygen tensions. Both alginate encapsulation and reduced oxygen tension significantly upregulated collagen II and aggrecan core protein expression (differentiation markers). In fact, after 4 weeks in alginate at 5% oxygen, differentiated gene expression was comparable to primary chondrocytes. Collagen I expression (dedifferentiation marker) decreased dramatically after alginate entrapment, while reduced oxygen tension had no effect. It is concluded that alginate encapsulation and reduced oxygen tension help restore key differentiated phenotypic markers of passaged chondrocytes. These findings have important implications for cartilage tissue engineering, since they enable the increase in differentiated cell numbers needed for the in vitro development of functional cartilaginous tissue suitable for implantation.

The objective of this study was to evaluate the biomechanical properties of newly formed cartilaginous tissue synthesized from isolated chondrocytes. Cartilage from articular joints of lambs was either digested in collagenase to isolated chondrocytes or cut into discs that were devitalized by multiple freeze-thaw cycles. Isolated cells were incubated in suspension culture in the presence of devitalized cartilage matrix for 3 weeks. Multiple chondrocyte/matrix constructs were assembled with fibrin glue and implanted subcutaneously in nude mice for up to 6 weeks. Testing methods were devised to quantify integration of cartilage pieces and mechanical properties of constructs. These studies showed monotonic increase with time in tensile strength, fracture strain, fracture energy, and tensile modulus to values 5-10% of normal articular cartilage by 6 weeks in vivo. Histological analysis indicated that chondrocytes grown on dead cartilage matrix produced new matrix that integrated individual cartilage pieces with mechanically functional tissue.

Chondrocytes have been generated in vitro from a range of progenitor cell types and by a number of strategies. However, achieving reconstitution of actual physiologically relevant, appropriately-laminated cartilage in situ that would be applicable to conditions, such as arthritis and cartilage degeneration remains elusive. This lack of success is multifactorial and includes limited cell source, decreased proliferation rate of mature chondrocytes, lack of maintenance of phenotype, reduced matrix synthesis, and poor integration with host tissue. We report an efficient approach for deriving mesenchymal chondroprogenitor cells from human embryonic stem cells. These cells generated tissue containing cartilage-specific matrix proteins that integrated in situ in a partial-thickness defect in ex vivo articular cartilage harvested from human arthritic joints. Given that stem cells provide a virtually inexhaustible supply of starting material and that our technique is easily scalable, cartilaginous tissue primed and grafted in this manner could be suitable for clinical translation.