Tissue engineering of human cartilage and osteochondral composites using recirculation bioreactors

Chondrocytes isolated from human foetal epiphyseal cartilage were seeded dynamically into polyglycolic acid (PGA) scaffolds and cultured in recirculation column bioreactors to produce tissue-engineered cartilage. Several culture techniques with the potential to provide endogenous growth factors and other conditions beneficial for de novo cartilage synthesis were investigated. Osteochondral composite constructs were generated by seeding separate PGA scaffolds with either foetal chondrocytes or foetal osteoblasts then suturing the scaffolds together before bioreactor cultivation. This type of co-culture system provided direct contact between the tissue-engineered cartilage and developing tissue-engineered bone and yielded significant improvements in cartilage quality. In the cartilage section of the composites, the concentrations of glycosaminoglycan (GAG) and total collagen were increased by 55% and 2.5-fold, respectively, compared with control cartilage cultures, while levels of collagen type II were similar to those in the controls. The osteochondral composites were harvested from the bioreactors as single units with good integration between the cartilage and bone tissues. Only the cartilage layer contained GAG while only the bone layer was mineralised. In other experiments, co-culture of tissue-engineered cartilage with pieces of ex-vivo cartilage or ex-vivo bone did not improve the quality of the cartilage relative to control cultures. Addition of 10(-6) M diacerein to the culture medium also had no effect on the properties of engineered cartilage. This work demonstrates the beneficial effects of generating cartilage tissues in contact with developing bone. It also demonstrates the feasibility of producing composite osteochondral constructs for clinical application using recirculation column bioreactors.     

Hydrogel optimization for cultured elastic chondrocytes seeded onto a polyglycolic acid scaffold

The purpose of this study was to compare the effect of different hydrogels on the production of tissue-engineered cartilage based on polyglycolic acid (PGA). Chondrocytes were isolated from adult sheep auricles. Alginate, Type I collagen, methylcellulose, and pluronic F127 hydrogels were evaluated, as were controls prepared without hydrogels. Proliferated chondrocytes were mixed with each hydrogel at 20 x 10(6) cells/mL and seeded onto PGA (1 x 1 x 0.2 cm, n = 60). The constructs were cultured with serum-free medium containing 5 ng/mL TGF-beta(2) and 5 ng/mL des(1-3)IGF-I in rotational bioreactors for up to 6 weeks. The cellular morphology, histology, and biochemistry were analyzed. Type I collagen, methylcellulose, and pluronic F127 displayed improved cartilage matrix deposition in terms of histology and biochemistry compared to alginate. It was not concluded that the combined seeding of chondrocytes and hydrogels on a PGA scaffold had significantly better effects than cell seeding without hydrogels. However, the histology and other useful findings in this ECM analyses suggested that Type I collagen and MC hydrogels were the best candidates for cartilage regeneration, because of their stimulation for chondrocyte proliferation in a three-dimensional culture as well as cartilage regeneration.     

Differential effects of growth factors on tissue-engineered cartilage

The effects of four regulatory factors on tissue-engineered cartilage were examined with specific focus on the ability to increase construct growth rate and concentrations of glycosaminoglycans (GAG) and collagen, the major extracellular matrix (ECM) components. Bovine calf articular chondrocytes were seeded onto biodegradable polyglycolic acid (PGA) scaffolds and cultured in medium with or without supplemental insulin-like growth factor (IGF-I), interleukin-4 (IL-4), transforming growth factor-beta1 (TGF-beta1) or platelet-derived growth factor (PDGF). IGF-I, IL-4, and TGF-beta1 increased construct wet weights by 1.5-2.9-fold over 4 weeks of culture and increased amounts of cartilaginous ECM components. IGF-I (10-300 ng/mL) maintained wet weight fractions of GAG in constructs seeded at high cell density and increased by up to fivefold GAG fractions in constructs seeded at lower cell density. TGF-beta1 (30 ng/mL) increased wet weight fractions of total collagen by up to 1.4-fold while maintaining a high fraction of type II collagen (79 plus minus 11% of the total collagen). IL-4 (1-100 ng/mL) minimized the thickness of the GAG-depleted region at the construct surfaces. PDGF (1-100 ng/mL) decreased construct growth rate and ECM fractions. Different regulatory factors thus elicit significantly different chondrogenic responses and can be used to selectively control the growth rate and improve the composition of engineered cartilage.     

Effects of Initial Cell Seeding Density for the Tissue Engineering of the Temporomandibular Joint Disc

Tissue engineering may provide a better treatment modality for postoperative discectomy patients. The TMJ disc is an ideal candidate for tissue engineering approaches because of its lack of an intrinsic regenerative ability. Unfortunately, basic knowledge related to TMJ disc tissue engineering is still at an infancy level and not on par to that related to articular cartilage tissue engineering. The objective of this study was to examine the effects of initial cell density of TMJ disc cells seeded in nonwoven poly-glycolic acid (PGA) scaffolds on the biochemical and biomechanical properties of constructs examined at 0, 3, and 6 weeks after seeding. Low, medium, and high seeding densities were chosen to be 15, 30, and 120 million cells per ml of scaffold, which were seeded using a spinner flask. Significant differences were found temporally and as a function of seeding density in morphology, total collagen, GAG content, and permeability of the constructs, but not in aggregate modulus. The high seeding density group outperformed the low and medium groups in collagen and GAG content at all time points measured. The high-density group produced a total of 55.37 ± 3.56 μg of collagen per construct, maintained 15.77 ± 1.86 μg of GAG per construct, and only shrunk to 50% of the original scaffold size. Permeability of the constructs at 6 weeks was decreased by 70% compared to 0 weeks.     

A novel simulation model for engineered cartilage growth in static systems

A novel mathematical model to simulate the growth of engineered cartilage in static systems is proposed. This model is based on material balances for the involved species (glycosaminoglycan and collagen, both pertaining to extracellular matrix), as well as mass-structured population balance for simulating cell growth and its proliferation within the scaffold. This model may simulate tissue growth on static culture taking place in Petri dishes, static flasks, and well plates for different types of scaffolds (i.e., poly(glycolic acid) [PGA], PGA/poly(l-lactic acid), and collagen sponge). This work aimed to demonstrate that the model approach proposed in previous works, regarding engineered cartilage growth on PGA scaffolds performed in rotating bioreactors, may also be applied to different scaffolds and system configurations. In particular, the balance equation for simulating collagen production is introduced, as well as the use of spatial averaging over the spatial region to compare experimental data with the model. Experimental data from the literature in terms of cells, glycosaminoglycans, and collagen content have been successfully compared with model results, thus demonstrating the validity of the proposed model, as well as its predictive capability.     

Effects of hedgehog proteins on tissue engineering of cartilage in vitro

The effects of three derivatives of the N-terminal signaling domain of hedgehog proteins on cartilage engineered in vitro were investigated, with specific focus on the ability to increase tissue growth rate and concentrations of major extracellular matrix components, that is, glycosaminoglycans (GAG) and collagen, and on the effects on morphological appearance of the tissue. Bovine articular chondrocytes were cultured on biodegradable polyglycolic acid (PGA) scaffolds with or without the addition of dipalmitoylated sonic hedgehog (dp-shh), dipalmitoylated indian hedgehog (dp-ihh), or sonic hedgehog dimer (shh-dimer) to medium with either 1% or 10% fetal bovine serum (FBS). All three hedgehog proteins dose-dependently increased construct weights (by up to 1.95-fold, dp-shh at 1,000 ng/mL) and the fraction of GAG over 4 weeks (by up to 2.7-fold, dp-shh at 1,000 ng/mL), as compared to control constructs. Dp-shh and dp-ihh elicited similar responses; a 10-fold higher concentration of nonacylated shh-dimer was necessary to reach comparable results. Positive hedgehog effects were more pronounced in medium containing 1% FBS than in medium containing 10% FBS; however, at either FBS concentration, cartilaginous tissues grown in the presence of hedgehog proteins appeared morphologically more mature. Hedgehog derivatives thus appear as promising candidates to improve the development and composition of engineered cartilage.     

Effect of low oxygen tension on tissue-engineered cartilage construct development in the concentric cylinder bioreactor

Cartilage is exposed to low oxygen tension in vivo, suggesting culture in a low-oxygen environment as a strategy to enhance matrix deposition in tissue-engineered cartilage in vitro. To assess the effects of oxygen tension on cartilage matrix accumulation, porous polylactic acid constructs were dynamically seeded in a concentric cylinder bioreactor with bovine chondrocytes and cultured for 3 weeks at either 20 or 5% oxygen tension. Robust chondrocyte proliferation and matrix deposition were achieved. After 22 days in culture, constructs from bioreactors operated at either 20 or 5% oxygen saturation had similar chondrocyte densities and collagen content. During the first 12 days of culture, the matrix glycosaminoglycan (GAG) deposition rate was 19.5 x 10(-9) mg/cell per day at 5% oxygen tension and 65% greater than the matrix GAG deposition rate at 20% oxygen tension. After 22 days of bioreactor culture, constructs at 5% oxygen contained 4.5 +/- 0.3 mg of GAG per construct, nearly double the 2.5 +/- 0.2 mg of GAG per construct at 20% oxygen tension. These data demonstrate that culture in bioreactors at low oxygen tension favors the production and retention of GAG within cartilage matrix without adversely affecting chondrocyte proliferation or collagen deposition. Bioreactor studies such as these can identify conditions that enhance matrix accumulation and construct development for cartilage tissue engineering.     

Engineering of implantable cartilaginous structures from bone marrow-derived mesenchymal stem cells

Fabrication of implantable cartilaginous structures that could be secured in the joint defect could provide an alternative therapeutic approach to prosthetic joint replacement. Herein we explored the possibility of using biodegradable hydrogels in combination with a polyglycolic acid (PGA) scaffold to provide an environment propitious to mesenchymal stem cells (MSCs) chondrogenic differentiation.We examined the influence of type I collagen gel and alginate combined with PGA meshes on the extracellular matrix composition of tissue-engineered transplants. MSCs were isolated from young rabbits, expanded in monolayers, suspended in each hydrogel, and loaded on PGA scaffolds. All constructs (n=48) were cultured in serum-free medium containing transforming growth factor beta-1, under dynamic conditions in specially designed bioreactors for 3-6 weeks. All cell-polymer constructs had a white, shiny aspect, and retained their initial size and shape over the culture period. Their thickness increased substantially over time, and no shrinkage was observed. All specimens developed a hyalin-like extracellular matrix containing glycosaminoglycans (GAGs) and type II collagen, but significant differences were observed among the three different groups. In PGA/MSCs and collagen-PGA/MSCs constructs, the cell growth phase and the chondrogenic differentiation phase of MSCs occurred during the first 3 weeks. In alginate-PGA/MSCs constructs, cells remained round in the hydrogel and cartilage extracellular matrix deposition was delayed. However, at 6 weeks, alginate-PGA/MSCs constructs exhibited higher contents of GAGs and lower contents of type I collagen. These results suggest that the implied time for the transplantation of in vitro engineered constructs depends, among other factors, on the nature of the scaffold envisioned. In this study, we demonstrated that the use of a composite hydrogel-PGA scaffold supported the in vitro growth of implantable cartilaginous structures cultured in a bioreactor system.     

Cell-nanofiber-based cartilage tissue engineering using improved cell seeding, growth factor, and bioreactor technologies

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-1 and transforming growth factor-beta1 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. Bioreactor-grown 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.     

Extracellular matrix protein gene expression of bovine chondrocytes cultured on resorbable scaffolds

It has been demonstrated that using cultured chondrocytes that have been seeded onto various biomatrices can enhance the quality of the articular cartilage repair tissue. As tissue-engineering becomes increasingly more complex there is a need to understand how a specific biomaterial may influence gene expression. In this study several commonly used scaffold materials for cartilage tissue engineering were evaluated with respect to their influence on matrix gene expression. Primary cultures of bovine chondrocytes were established in monolayer then seeded onto polylactic acid (PLLA), polyglycolic acid (PGA), collagen matrices. The induction of collagen type I, collagen type II, and aggrecan was observed at various time points on these biomaterials using RT-PCR. The collagen type I gene was upregulated on collagen scaffolds throughout the culture period. PLLA and PGA showed initial induction followed by downregulation. Monolayer culture did not induce collagen I message. Collagen II genes were selectively upregulated after 72 and 96 h post seeding depending the scaffold material. Monolayer culture had strong induction of collagen II. The aggrecan protein was consistently expressed in all scaffold materials cultures and monolayer.     

Oscillatory perfusion seeding and culturing of osteoblast-like cells on porous beta-tricalcium phosphate scaffolds

Perfusion culture systems have proven to be effective bioreactors for constructing tissue engineered bone in vitro, but existing circuit-based perfusion systems are complicated and costly for conditioned culture due to the large medium volume required. A compact perfusion system for artificial bone fabrication using oscillatory flow is described here. Mouse osteoblast-like MC 3T3-E1 cells were seeded at 1.5 x 10(6) cells/100 microL and cultured for 6 days in porous ceramic beta-tricalcium phosphate scaffolds (10 mm in diameter, 8 mm in height) by only 1.5 mL culture media per scaffold. The seeding efficiency, cell proliferation, distribution and viability, and promotion of early osteogenesis by both a static and an oscillatory perfusion method were evaluated. The oscillatory perfusion method generated higher seeding efficiency, alkaline phosphatase activity, and scaffold cellularity (by DNA content) after 6 days of culture. Stereomicroscopic observation of 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staining and Calcein-AM/propidium iodide double staining also demonstrated homogeneous seeding, proliferation, and viability of cells throughout the scaffolds in the oscillatory perfusion system. By contrast, the static culture yielded polarized seeding and proliferation favoring the outer and upper scaffold surfaces, with only dead cells in the center of the scaffolds. Thus, these results suggest that the oscillatory flow condition not only allow a better seeding efficiency and homogeneity, but also facilitates uniform culture and early osteogenic differentiation. The oscillatory perfusion system could be a simple and effective bioreactor for bone tissue engineering.     

Polymer scaffolds fabricated with pore-size gradients as a model for studying the zonal organization within tissue-engineered cartilage constructs

The zonal organization of cells and extracellular matrix (ECM) constituents within articular cartilage is important for its biomechanical function in diarthroidal joints. Tissue-engineering strategies adopting porous three-dimensional (3D) scaffolds offer significant promise for the repair of articular cartilage defects, yet few approaches have accounted for the zonal structural organization as in native articular cartilage. In this study, the ability of anisotropic pore architectures to influence the zonal organization of chondrocytes and ECM components was investigated. Using a novel 3D fiber deposition (3DF) technique, we designed and produced 100% interconnecting scaffolds containing either homogeneously spaced pores (fiber spacing, 1 mm; pore size, about 680 microm in diameter) or pore-size gradients (fiber spacing, 0.5-2.0 mm; pore size range, about 200-1650 microm in diameter), but with similar overall porosity (about 80%) and volume fraction available for cell attachment and ECM formation. In vitro cell seeding showed that pore-size gradients promoted anisotropic cell distribution like that in the superficial, middle, and lower zones of immature bovine articular cartilage, irrespective of dynamic or static seeding methods. There was a direct correlation between zonal scaffold volume fraction and both DNA and glycosaminoglycan (GAG) content. Prolonged tissue culture in vitro showed similar inhomogeneous distributions of zonal GAG and collagen type II accumulation but not of GAG:DNA content, and levels were an order of magnitude less than in native cartilage. In this model system, we illustrated how scaffold design and novel processing techniques can be used to develop anisotropic pore architectures for instructing zonal cell and tissue distribution in tissue-engineered cartilage constructs.     

In vitro chondrocyte behavior on porous biodegradable poly (e-caprolactone)/polyglycolic acid scaffolds for articular chondrocyte adhesion and proliferation

In this study, poly(e-caprolactone)/polyglycolic acid (PCL/PGA) scaffolds for repairing articular cartilage were fabricated via solid-state cryomilling along with compression molding and porogen leaching. Four distinct scaffolds were fabricated using this approach by four independent cryomilling times. These scaffolds were assessed for their suitability to promote articular cartilage regeneration with in vitro chondrocyte cell culture studies. The scaffolds were characterized for pore size, porosity, swelling ratio, compressive, and thermal properties. Cryomilling time proved to significantly affect the physical, mechanical, and morphological properties of the scaffolds. In vitro bovine chondrocyte culture was performed dynamically for 1, 7, 14, 28, and 35 days. Chondrocyte viability and adhesion were tested using MTT assay and scanning electron microscopy micrographs. Glycosaminoglycan (GAG) and DNA assays were performed to investigate the extracellular matrix (ECM) formation and cell proliferation, respectively. PCL/PGA scaffolds demonstrated high porosity for all scaffold types. Morphological analysis and poly(ethylene oxide) continuity demonstrated the existence of a co-continuous network of interconnected pores with pore sizes appropriate for tissue engineering and chondrocyte ingrowth. While mean pore size decreased, water uptake and compressive properties increased with increasing cryomilling times. Compressive modulus of 12, 30, and 60 min scaffolds matched the compressive modulus of human articular cartilage. Viable cells increased besides increase in cell proliferation and ECM formation with progress in culture period. Chondrocytes exhibited spherical morphology on all scaffold types. The pore size of the scaffold affected chondrocyte adhesion, proliferation, and GAG secretion. The results indicated that the 12 min scaffolds delivered promising results for applications in articular cartilage repair.     

Evaluation of biodegradable, three-dimensional matrices for tissue engineering of heart valves

A crucial factor in tissue engineering of heart valves is the type of scaffold material. In the following study, we tested three different biodegradable scaffold materials, polyglycolic acid (PGA), polyhydroxyalkanoate (PHA), and poly-4-hydroxybutyrate (P4HB), as scaffolds for tissue engineering of heart valves. We modified PHA and P4HB by a salt leaching technique to create a porous matrix. We constructed trileaflet heart valve scaffolds from each polymer and tested them in a pulsatile flow bioreactor. In addition, we evaluated the cell attachment to our polymers by creating four tubes of each material (length equals 4 cm; inner diameter, 0.5 cm), seeding each sample with 8,000,000 ovine vascular cells, and incubating the cell-polymer construct for 8 days (37 degrees C and 5% CO2). The seeded vascular constructs were exposed to continuous flow for 1 hour. Analysis of samples included DNA assay before and after flow exposure, 4-hydroxyproline assay, and environmental scanning electron microscopy (ESEM). We fabricated trileaflet heart valve scaffolds from porous PHA and porous P4HB, which opened and closed synchronously in a pulsatile bioreactor. It was not possible to create a functional trileaflet heart valve scaffold from PGA. After seeding and incubating the PGA-, PHA-, and P4HB-tubes, there were significantly (p < 0.001) more cells on PGA compared with PHA and P4HB. There were no significant differences among the materials after flow exposure, but there was a significantly higher collagen content (p < 0.017) on the PGA samples compared with P4HB and PHA. Cell attachment and collagen content was significantly higher on PGA samples compared with PHA and P4HB. However, PHA and P4HB also demonstrate a considerable amount of cell attachment and collagen development and share the major advantage that both materials are thermoplastic, making it possible to mold them into the shape of a functional scaffold for tissue engineering of heart valves.     

The effect of type II collagen coating of chitosan fibrous scaffolds on mesenchymal stem cell adhesion and chondrogenesis

The biocompatibility of chitosan and its similarity to glycosaminoglycans (GAG) make it attractive for cartilage tissue engineering. We have previously reported improved chondrogenesis but limited cell adhesion on chitosan scaffolds. Our objectives were to produce chitosan scaffolds coated with different densities of type II collagen and to evaluate the effect of this coating on mesenchymal stem cell (MSC) adhesion and chondrogenesis.Chitosan fibrous scaffolds were obtained by a wet spinning method and coated with type II collagen at two different densities. A polyglycolic acid mesh served as a reference group. The scaffolds were characterized by Fourier-transform infrared spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and type II collagen content. Constructs were analyzed after MSCs seeding via live/dead assay, weight and DNA evaluations, SEM, and TEM. Constructs were cultured in chondrogenic medium for 21 days prior to quantitative analysis (weight, DNA, and GAG), SEM, TEM, histology, immunohistochemistry, and quantitative real time polymerase chain reaction. The cell attachment and distribution after seeding correlated with the density of type II collagen. The cell number, the matrix production, and the expression of genes specific for chondrogenesis were improved after culture in collagen coated chitosan constructs.These findings encourage the use of type II collagen for coating chitosan scaffolds to improve MSCs adhesion and chondrogenesis, and confirm the importance of biomimetic scaffolds for tissue engineering.     

Chitosan scaffolds: interconnective pore size and cartilage engineering

This study was designed to determine the effect of interconnective pore size on chondrocyte proliferation and function within chitosan sponges, and compare the potential of chitosan and polyglycolic acid (PGA) matrices for chondrogenesis. Six million porcine chondrocytes were seeded on each of 52 prewetted scaffolds consisting of chitosan sponges with (1) pores 10 microm in diameter (n=10, where n is the number of samples); (2) pores measuring 10-50 microm in diameter (n=10); and (3) pores measuring 70-120 microm in diameter (n=10), versus (4) polyglycolic acid mesh (n=22), as a positive control. Constructs were cultured for 28 days in a rotating bioreactor prior to scanning electron microscopy (SEM), histology, and determination of their water, DNA, glycosaminoglycan (GAG) and collagen II contents. Parametric data was compared (p=0.05) with an ANOVA and Tukey's Studentized range test. PGA constructs consisted essentially of a matrix containing more cells than normal cartilage. Whereas very few remnants of PGA remained, chitosan scaffolds appeared intact. DNA and GAG concentrations were greater in PGA scaffolds than in any of the chitosan groups. However, chitosan sponges with the largest pores contained more chondrocytes, collagen II and GAG than the matrix with the smallest pores. Constructs produced with PGA contained less water and more GAG than all chitosan groups. Chondrocyte proliferation and metabolic activity improved with increasing interconnective pore size of chitosan matrices. In vitro chondrogenesis is possible with chitosan but the composition of constructs produced on PGA more closely approaches that of natural cartilage.     

Comparison of scaffolds and culture conditions for tissue engineering of the knee meniscus

The menisci of the knee are semilunar fibrocartilaginous structures critical in load bearing, shock absorption, stability, and lubrication. In this study, two commonly used biomaterials, a hydrogel (agarose) and a nonwoven mesh polymer [poly(glycolic acid); PGA], were compared for suitability as scaffold materials for tissue engineering the knee meniscus. In addition, a rotating wall bioreactor culture of both scaffold materials was compared with static cultures. Constructs were cultured for up to 7 weeks in static and rotating wall bioreactor culture. Cell numbers were 22 times higher in PGA than agarose after 7 weeks in culture. Static PGA scaffolds had more than twice the amount of sulfated glycosaminoglycans and three times the amount of collagen compared to static agarose constructs at week 7. The rotating wall bioreactor was not found with increase matrix production or cell proliferation significantly over static cultures.     

Bone and cartilage tissue constructs grown using human bone marrow stromal cells, silk scaffolds and rotating bioreactors

Human bone marrow contains a population of bone marrow stromal cells (hBMSCs) capable of forming several types of mesenchymal tissues, including bone and cartilage. The present study was designed to test whether large cartilaginous and bone-like tissue constructs can be selectively engineered using the same cell population (hBMSCs), the same scaffold type (porous silk) and same hydrodynamic environment (construct settling in rotating bioreactors), by varying the medium composition (chondrogenic vs. osteogenic differentiation factors). The hBMSCs were harvested, expanded and characterized with respect to their differentiation potential and population distribution. Passage two cells were seeded on scaffolds and cultured for 5 weeks in bioreactors using osteogenic, chondrogenic or control medium. The three media yielded constructs with comparable wet weights and compressive moduli (25 kPa). Chondrogenic medium yielded constructs with higher amounts of DNA (1.5-fold) and glycosaminoglycans (GAG, 4-fold) per unit wet weight (ww) than control medium. In contrast, osteogenic medium yielded constructs with higher dry weight (1.6-fold), alkaline phosphatase (AP) activity (8-fold) and calcium content (100-fold) per unit ww than control medium. Chondrogenic medium yielded constructs that were weakly positive for GAG by contrast-enhanced MRI and alcian blue stain, whereas osteogenic medium yielded constructs that were highly mineralized by μCT and von Kossa stain. Engineered bone constructs were large (8 mm diameter × 2 mm thick disks) and resembled trabecular bone with respect to structure and mineralized tissue volume fraction (12%).     

Characterization of polylactic acid-polyglycolic acid composites for cartilage tissue engineering

The objective of this study was to determine the effects of scaffold composition on the physical properties, adhesion, and growth of bovine articular chondrocytes on polylactic acid (PLA)/polyglycolic acid (PGA) composites. Nonwoven meshes of PGA were coated with PLA, using a solvent evaporation technique that resulted in composites with fractional PLA contents ranging from 0 to 68%. The compressive modulus of scaffolds increased linearly with the addition of PLA, ranging from less than 1 kPa for PGA to approximately 20 kPa for scaffolds with 68% PLA content. The characteristic degradation time of these scaffolds also increased from approximately 5 days for 0% PLA to 45 days for 68% PLA. Addition of PLA decreased cell seeding efficiency from 48% for 0% PLA scaffolds to 27% for 68% PLA scaffolds. Cells seeded onto 27% PLA scaffolds increased 3-fold in number over 4 weeks in culture, whereas cells seeded onto 68% PLA increased only 2-fold in number. Scanning electron microscopy indicated that cells attached to PGA appeared flat with many small processes, whereas those attached to PLA were more rounded. These studies provide important information for the design of scaffolds for cartilage tissue engineering.     

Low-density cultures of bovine chondrocytes: effects of scaffold material and culture system

Chondrocytes were seeded on either agarose or polyglycolic acid (PGA) unwoven meshes at 10 million cells/ml of scaffold volume to evaluate the effect that these two biomaterials have on the low-density culture of chondrocytes in a rotating-wall bioreactor. For both static and bioreactor culture, agarose constructs contained more glycosaminoglycan than their PGA counterparts. However, the PGA constructs contained more collagen for both culture conditions when compared to agarose. For the low seeding density of this study, PGA constructs cultured in the bioreactor did not outperform static cultures when comparing collagen content after 8 weeks. The mechanical properties of the PGA constructs also did not improve with culture time. Similar results were observed with the agarose culture, though both static- and bioreactor-culture agarose constructs exhibited increases in aggregate modulus at the end of the culture period. As in PGA culture, chondrocytes cultured in agarose may require a higher density to reap the benefits of the bioreactor environment.