Scaffolds In Bioreactor Research Articles

Influence of different pressure regimes on the properties of an engineered small-diameter vascular scaffold tested in a custom-made bioreactor.

Vascular tissue engineering endeavors to design, fabricate, and validate biodegradable and bioabsorbable small-diameter vascular scaffolds engineered with bioactive molecules, capable of meeting the challenges imposed by commercial vascular prostheses. A comprehensive investigation of these engineered scaffolds in bioreactor is deemed essential as a prerequisite before any in vivo experimentation in order to get information regarding their behavior under physiological conditions and predict the biological activities they will possess. This study focuses on an innovative electrospun scaffold made of poly(caprolactone) and poly(glycerol sebacate), integrating quercetin, able to modulate inflammation, and gelatin, necessary to reduce permeability. A custom-made bioreactor was used to assess the performances of the scaffolds maintained under different pressure regimes, covering the human physiological pressure range. As results, the 3D microfibrous architecture was notably influenced by the release of bioactives, maintaining the adequate properties needed for the in vivo regeneration and scaffolds showed mechanical properties similar to human native artery. Release of gelatin was adequate to avoid blood leakage and useful to make the material porous during the testing period, whereas the amount of released quercetin was useful to counteract the post-surgery inflammation. This study showcases the successful validation of an engineered scaffold in a bioreactor, enabling to consider it as a promising candidate for vascular substitutes in in vivo applications. Our approach represents a significant leap forward in the field of vascular tissue engineering, offering a multifaceted solution to the complex challenges associated with small-diameter vascular prostheses.&#xD.

Osteogenic Activity on NaOH-Etched Three-Dimensional-Printed Poly-ɛ-Caprolactone Scaffolds in Perfusion or Spinner Flask Bioreactor.

Bioreactor systems, for example, spinner flask and perfusion bioreactors, and cell-seeded three-dimensional (3D)-printed scaffolds are used in bone tissue engineering strategies to stimulate cells and produce bone tissue suitable for implantation into the patient. The construction of functional and clinically relevant bone graft using cell-seeded 3D-printed scaffolds within bioreactor systems is still a challenge. Bioreactor parameters, for example, fluid shear stress and nutrient transport, will crucially affect cell function on 3D-printed scaffolds. Therefore, fluid shear stress induced by spinner flask and perfusion bioreactors might differentially affect osteogenic responsiveness of pre-osteoblasts inside 3D-printed scaffolds. We designed and fabricated surface-modified 3D-printed poly-ɛ-caprolactone (PCL) scaffolds, as well as static, spinner flask, and perfusion bioreactors to determine fluid shear stress and osteogenic responsiveness of MC3T3-E1 pre-osteoblasts seeded on the scaffolds in the bioreactors using finite element (FE)-modeling and experiments. FE-modeling was used to quantify wall shear stress (WSS) distribution and magnitude inside 3D-printed PCL scaffolds within spinner flask and perfusion bioreactors. MC3T3-E1 pre-osteoblasts were seeded on NaOH surface-modified 3D-printed PCL scaffolds, and cultured in customized static, spinner flask, and perfusion bioreactors up to 7 days. The scaffolds' physicochemical properties and pre-osteoblast function were assessed experimentally. FE-modeling showed that spinner flask and perfusion bioreactors locally affected WSS distribution and magnitude inside the scaffolds. The WSS distribution was more homogeneous inside scaffolds in perfusion than in spinner flask bioreactors. The average WSS on scaffold-strand surfaces ranged from 0 to 6.5 mPa for spinner flask bioreactors, and from 0 to 4.1 mPa for perfusion bioreactors. Surface modification of scaffolds by NaOH resulted in a surface with a honeycomb-like pattern and increased surface roughness (1.6-fold), but decreased water contact angle (0.3-fold). Both spinner flask and perfusion bioreactors increased cell spreading, proliferation, and distribution throughout the scaffolds. Perfusion, but not spinner flask bioreactors more strongly enhanced collagen (2.2-fold) and calcium deposition (2.1-fold) throughout the scaffolds after 7 days compared with static bioreactors, likely due to uniform WSS-induced mechanical stimulation of the cells revealed by FE-modeling. In conclusion, our findings indicate the importance of using accurate FE models to estimate WSS and determine experimental conditions for designing cell-seeded 3D-printed scaffolds in bioreactor systems. Impact Statement The success of cell-seeded three-dimensional (3D)-printed scaffolds depends on cell stimulation by biomechanical/biochemical factors to produce bone tissue suitable for implantation into the patient. We designed and fabricated surface-modified 3D-printed poly-ɛ-caprolactone (PCL) scaffolds, as well as static, spinner flask, and perfusion bioreactors to determine wall shear stress (WSS) and osteogenic responsiveness of pre-osteoblasts seeded on the scaffolds using finite element (FE)-modeling and experiments. We found that cell-seeded 3D-printed PCL scaffolds within perfusion bioreactors more strongly enhanced osteogenic activity than within spinner flask bioreactors. Our results indicate the importance of using accurate FE-models to estimate WSS and determine experimental conditions for designing cell-seeded 3D-printed scaffolds in bioreactor systems.

Time-lapsed imaging of nanocomposite scaffolds reveals increased bone formation in dynamic compression bioreactors

Progress in bone scaffold development relies on cost-intensive and hardly scalable animal studies. In contrast to in vivo, in vitro studies are often conducted in the absence of dynamic compression. Here, we present an in vitro dynamic compression bioreactor approach to monitor bone formation in scaffolds under cyclic loading. A biopolymer was processed into mechanically competent bone scaffolds that incorporate a high-volume content of ultrasonically treated hydroxyapatite or a mixture with barium titanate nanoparticles. After seeding with human bone marrow stromal cells, time-lapsed imaging of scaffolds in bioreactors revealed increased bone formation in hydroxyapatite scaffolds under cyclic loading. This stimulatory effect was even more pronounced in scaffolds containing a mixture of barium titanate and hydroxyapatite and corroborated by immunohistological staining. Therefore, by combining mechanical loading and time-lapsed imaging, this in vitro bioreactor strategy may potentially accelerate development of engineered bone scaffolds and reduce the use of animals for experimentation.

Fabrication and in vitro evaluation of a packed-bed bioreactor based on galactosylated poly(ethylene terephthalate) microfibrous scaffolds

Bioartificial liver (BAL) comprising a functional bioreactor represents a promising treatment, bridging patients with acute liver failure (ALF) to liver transplantation. In this research, a packed-bed (PB) bioreactor by utilizing galactosylated poly(ethylene terephthalate) microfibrous carrier (PET-Gal) was fabricated and evaluated in vitro. Human induced hepatocytes (hiHeps) were dynamically seeded into a PET-Gal loaded bioreactor and cultured for 10 days. During perfusion culture, the effects of bioreactor scaffold on hepatocyte seeding, growth and functionality (albumin secretion and urea production) were determined, compared with that of control with non-modified PET carrier. Afterwards, drug metabolism and detoxification ability of hiHeps were measured. Results demonstrated that the seeding rate and proliferation fold within PET-Gal loaded bioreactor were significantly higher than that of control, resulting in a cell density of (8.17 ± 0.52)×107 cells/cm3. In contrast to flattened morphology observed in the control, hiHeps cultured in PET-Gal based bioreactor displayed as three-dimensional (3D) cell aggregates with close cell-matrix and cell-cell interactions and high cell viability, leading to higher albumin secretion and urea production. Besides, there were high levels of phase I drug metabolism and ammonia elimination for PET-Gal cultured cells. Therefore, the PET-Gal based PB bioreactor fabricated herein supports high-density hepatocyte growth and meanwhile preserves hepatic functionality, showing great potential in BAL systems application.

Stimulation of cell growth and neurogenesis using protein-functionalized microfibrous scaffolds and fluid flow in bioreactors

Microfibrous viscose-rayon scaffolds functionalized with poly-L-lysine and laminin were used to culture pheochromocytoma (PC12) cells in recirculation bioreactors under bulk laminar flow conditions. To determine the effects of continuous forced convective flow on cell viability, growth, and neurogenesis, the cultures were exposed to fluid-induced shear stresses of magnitude 2.7 × 10–5 to 4.6 × 10–3 Pa for up to 12 days. The results were compared with those obtained using static scaffold and static monolayer (2D) control cultures. Cell growth rates were 1.5- to 1.8-fold greater in the bioreactor scaffold cultures relative to the controls without flow. After treatment of the cells with nerve growth factor, development and extension of neurite networks and expression of several neural differentiation markers including β3-tubulin, shootin1, and EphA2 were also substantially enhanced in the presence of fluid convection. In the control cultures without fluid flow, cell growth and differentiation were greater in the static scaffold cultures than in the static 2D monolayers. This work demonstrates the significant beneficial effects of features of the physical culture environment, such as hydrodynamic conditions and cell attachment surface geometry, in regulating culture performance. Flow devices capable of supporting long-term cell culture while delivering controlled levels of hydrodynamic shear are a valuable practical tool for promoting nerve tissue development and regeneration in vitro.

Expansion of Bone Marrow Mesenchymal Stromal Cells in Perfused 3D Ceramic Scaffolds Enhances In Vivo Bone Formation.

Bone marrow-derived mesenchymal stromal cells (BMSC), when expanded directly within 3D ceramic scaffolds in perfusion bioreactors, more reproducibly form bone when implanted in vivo as compared to conventional expansion on 2D polystyrene dishes/flasks. Since the bioreactor-based expansion on 3D ceramic scaffolds encompasses multiple aspects that are inherently different from expansion on 2D polystyrene, we aimed to decouple the effects of specific parameters among these two model systems. We assessed the effects of the: 1) 3D scaffold vs. 2D surface; 2) ceramic vs. polystyrene materials; and 3) BMSC niche established within the ceramic pores during in vitro culture, on subsequent in vivo bone formation. While BMSC expanded on 3D polystyrene scaffolds in the bioreactor could maintain their in vivo osteogenic potential, results were similar as BMSC expanded in monolayer on 2D polystyrene, suggesting little influence of the scaffold 3D environment. Bone formation was most reproducible when BMSC are expanded on 3D ceramic, highlighting the influence of the ceramic substrate. The presence of a pre-formed niche within the scaffold pores had negligible effects on the in vivo bone formation. The results of this study allow a greater understanding of the parameters required for perfusion bioreactor-based manufacturing of osteogenic grafts for clinical applications.

Reconstruction of large mandibular defects using autologous tissues generated from in vivo bioreactors.

A significant clinical challenge in craniofacial surgery is the reconstruction of large mandibular defects. In this work, we demonstrated that vascularized tissues of large volume and custom geometry can be generated from in vivo bioreactors implanted against the rib periosteum in an ovine model. The effects of different bioreactor scaffold material on tissue ingrowth were measured. To minimize donor site morbidity, tissues generated from bioreactors filled with synthetic graft were transferred as either vascularized free flaps or avascular grafts to a large mandibular defect. It was demonstrated that synthetic graft in an in vivo bioreactor is sufficient to produce free tissue bone flaps capable of integrating with native tissues when transferred to a large mandibular defect in an ovine model.

Implantable tissue-engineered blood vessels from human induced pluripotent stem cells.

Derivation of functional vascular smooth muscle cells (VSMCs) from human induced pluripotent stem cells (hiPSCs) to generate tissue-engineered blood vessels (TEBVs) holds great potential in treating patients with vascular diseases. Herein, hiPSCs were differentiated into alpha-smooth muscle actin (α-SMA) and calponin-positive VSMCs, which were seeded onto polymer scaffolds in bioreactors for vascular tissue growth. A functional TEBV with abundant collagenous matrix and sound mechanics resulted, which contained cells largely positive for α-SMA and smooth muscle myosin heavy chain (SM-MHC). Moreover, when hiPSC-derived TEBV segments were implanted into nude rats as abdominal aorta interposition grafts, they remained unruptured and patent with active vascular remodeling, and showed no evidence of teratoma formation during a 2-week proof-of-principle study. Our studies represent the development of the first implantable TEBVs based on hiPSCs, and pave the way for developing autologous or allogeneic grafts for clinical use in patients with vascular disease.

Fluid flow and mass transfer over circular strands using the lattice Boltzmann method

Knowledge of the fluid flow and mass transfer over circular strands is fundamental to the cell culture of tissue scaffolds in bio-reactors. This paper presents a study on the simulation of fluid flow and mass transfer over the circular strands of a tissue scaffold by using the multiple relaxation time lattice Boltzmann method for the low Reynolds number regime, with Re D = 0.01 and 0.1, respectively. The mass transfer problem approximates the transport of a scalar nutrient from the bulk fluid to the strand surface, such as is encountered in the flow through tissue scaffolds placed in bio-reactors. The circular geometry of the scaffold strand is treated and implemented by means of the interpolated bounce-back boundary condition formulation. Our simulation illustrates that the flow accelerates around the strand, resulting in the maximum shear stress at the shoulder of the strand and that diffusion mass transfer plays the dominant role in the scalar transport. The local Sherwood number varies significantly over the surface of the strand, with a peak value located on the upstream surface. Increasing the Schmidt number of the scalar and decreasing the blockage ratio results in higher mass transfer rates on the surface of the stand. Overall, the simulation results provide one with the insight into the fluid flow and mass transfer over the circular strands of a tissue scaffold in a bio-reactor, which would be impractical to obtain by experiments.

Design and validation of a dynamic cell-culture system for bone biology research and exogenous tissue-engineering applications

Bone lacunocanalicular fluid flow ensures chemotransportation and provides a mechanical stimulus to cells. Traditional static cell-culture methods are ill-suited to study the intricacies of bone biology because they ignore the three-dimensionality of meaningful cellular networks and the lacunocanalicular system; furthermore, reliance on diffusion alone for nutrient supply and waste product removal effectively limits scaffolds to 2-3 mm thickness. In this project, a flow-perfusion system was custom-designed to overcome these limitations: eight adaptable chambers housed cylindrical cell-seeded scaffolds measuring 12 or 24 mm in diameter and 1-10 mm in thickness. The porous scaffolds were manufactured using a three-dimensional (3D) periodic microprinting process and were composed of hydroxyapatite/tricalcium phosphate with variable thicknesses, strut sizes, pore sizes and structural configurations. A multi-channel peristaltic pump drew medium from parallel reservoirs and perfused it through each scaffold at a programmable rate. Hermetically sealed valves permitted sampling or replacement of medium. A gas-permeable membrane allowed for gas exchange. Tubing was selected to withstand continuous perfusion for > 2 months without leakage. Computational modelling was performed to assess the adequacy of oxygen supply and the range of fluid shear stress in the bioreactor-scaffold system, using 12 × 6 mm scaffolds, and these models suggested scaffold design modifications that improved oxygen delivery while enhancing physiological shear stress. This system may prove useful in studying complex 3D bone biology and in developing strategies for engineering thick 3D bone constructs. Copyright © 2013 John Wiley & Sons, Ltd.

Modeling of the Flow within Scaffolds in Perfusion Bioreactors

Tissue engineering aims to produce artificial organs and tissues for transplant treatments, in which cultivating cells on scaffolds in bioreactors is of critical importance. To control the cultivating process, the knowledge of the fluid flow inside and around a scaffold in the bioreactor is essential. However, due to the complicated microstructure of a scaffold, it is difficult, or even impossible, to gain such knowledge experimentally. In contrast, numerical methods employing computa- tional fluid dynamics (CFD) have proven promising to alleviate the problem. In this research the fluid flow in perfusion bioreactors is studied with numerical methods. The emphasis is on investigating the effect of the controllable parameters in both the scaffold fabrication (i.e., the diameter of scaffold strand and the distance between two strands) and cell culture process (i.e., the flow rate) on the distribution of shear stress within the scaffold in a perfusion bioreactor. The knowledge obtained in this study will allow for improved control strategies in scaffold fabrication and cell culturing experiments.

Engineering bone tissue from human embryonic stem cells

In extensive bone defects, tissue damage and hypoxia lead to cell death, resulting in slow and incomplete healing. Human embryonic stem cells (hESC) can give rise to all specialized lineages found in healthy bone and are therefore uniquely suited to aid regeneration of damaged bone. We show that the cultivation of hESC-derived mesenchymal progenitors on 3D osteoconductive scaffolds in bioreactors with medium perfusion leads to the formation of large and compact bone constructs. Notably, the implantation of engineered bone in immunodeficient mice for 8 wk resulted in the maintenance and maturation of bone matrix, without the formation of teratomas that is consistently observed when undifferentiated hESCs are implanted, alone or in bone scaffolds. Our study provides a proof of principle that tissue-engineering protocols can be successfully applied to hESC progenitors to grow bone grafts for use in basic and translational studies.

A miniaturized, optically accessible bioreactor for systematic 3D tissue engineering research

Perfusion bioreactors are widely used in tissue engineering and pharmaceutical research to provide reliable models of tissue growth under controlled conditions. Destructive assays are not able to follow the evolution of the growing tissue on the same construct, so it is necessary to adopt non-destructive analysis. We have developed a miniaturized, optically accessible bioreactor for interstitial perfusion of 3D cell-seeded scaffolds. The scaffold adopted was optically transparent, with highly defined architecture. Computational fluid dynamics (CFD) analysis was useful to predict the flow behavior in the bioreactor scaffold chamber (that was laminar flow, Re = 0.179, with mean velocity equal to 100 microns/s). Moreover, experimental characterization of the bioreactor performance gave that the maximum allowable pressure was 0.06MPa and allowable flow rate up to 25ml/min. A method, to estimate quantitatively and non destructively the cell proliferation (from 15 to 43 thousand cells) and tissue growth (from 2% to 43%) during culture time, was introduced and validated. An end point viability test was performed to check the experimental set-up overall suitability for cell culture with successful results. Morphological analysis was performed at the end time point to show the complex tridimensional pattern of the biological tissue growth. Our system, characterized by controlled conditions in a wide range of allowable flow rate and pressure, permits to systematically study the influence of several parameters on engineered tissue growth, using viable staining and a standard fluorescence microscope.

Adipose tissue as a stem cell source for musculoskeletal regeneration

Adipose tissue is an abundant, easily accessible, and reproducible cell source for musculo-skeletal regenerative medicine applications. Initial derivation steps yield a heterogeneous population of cells of stromal vascular fraction (SVF) cells. Subsequent adherent selection of the SVF results in a relatively homogeneous population of adipose-derived stromal/stem cells (ASCs) capable of adipogenic, chondrogenic, myogenic, and osteogenic differentiation in vitro on scaffolds in bioreactors and in vivo in pre-clinical animal models. Unlike hematopoietic cells, ASCs do not elicit a robust lymphocyte reaction and instead release immunosuppressive factors, such as prostaglandin E2. These immunomodulatory features suggest that allogeneic and autologous ASCs will engraft successfully for tissue regeneration purposes. The differentiation and expansion potential of ASCs can be modified by growth factors, bio-inductive scaffolds, and bioreactors providing environmental control and biophysical stimulation. Gene therapy approaches using lentiviral transduction can be used to direct differentiation of ASCs to particular lineages. We discuss the utility of ASCs for musculo-skeletal tissue repair and some of the technologies that can be implemented to unlock the full regenerative potential of these highly valuable cells.

Computational fluid dynamics modeling of momentum transport in rotating wall perfused bioreactor for cartilage tissue engineering

In this study, computational fluid dynamics (CFD) analysis of a rotating-wall perfused-vessel (RWPV) bioreactor is performed to characterize the complex hydrodynamic environment for the simulation of cartilage development in RWPV bioreactor in the presence of tissue-engineered cartilage constructs, i.e., cell-chitosan scaffolds. Shear stress exerted on chitosan scaffolds in bioreactor was calculated for different rotational velocities in the range of 33–38 rpm. According to the calculations, the lateral and lower surfaces were exposed to 0.07926–0.11069 dyne/cm 2 and 0.05974–0.08345 dyne/cm 2, respectively, while upper surfaces of constructs were exposed to 0.09196–0.12847 dyne/cm 2. Results validate adequate hydrodynamic environment for scaffolds in RWPV bioreactor for cartilage tissue development which concludes the suitability of operational conditions of RWPV bioreactor.

Optical Mapping of Impulse Propagation in Engineered Cardiac Tissue

Cardiac tissue engineering has a potential to provide functional, synchronously contractile tissue constructs for heart repair, and for studies of development and disease using in vivo-like yet controllable in vitro settings. In both cases, the utilization of bioreactors capable of providing biomimetic culture environments is instrumental for supporting cell differentiation and functional assembly. In the present study, neonatal rat heart cells were cultured on highly porous collagen scaffolds in bioreactors with electrical field stimulation. A hallmark of excitable tissues such as myocardium is the ability to propagate electrical impulses. We utilized the method of optical mapping to measure the electrical impulse propagation. The average conduction velocity recorded for the stimulated constructs (14.4 +/- 4.1 cm/s) was significantly higher than that of the nonstimulated constructs (8.6 +/- 2.3 cm/s, p = 0.003). The measured electrical propagation properties correlated to the contractile behavior and the compositions of tissue constructs. Electrical stimulation during culture significantly improved amplitude of contractions, tissue morphology, and connexin-43 expression compared to the nonsimulated controls. These data provide evidence that electrical stimulation during bioreactor cultivation can improve electrical signal propagation in engineered cardiac constructs.

Adipose tissue-derived stem cell expansion on collagen/chitosan scaffolds in bioreactor

Recent reports indicate that adipose tissue is a novel source of multipotent stem cells, which can be used in cell therapy and tissue engineering. However, the static amplification of adipose tissue-derived stem cells(ADSCs) is a time-consuming procedure and prone to contamination, and it's hard to meet the need of clinical applications. To obtain the stem cells in a great number of quantity and with stemness well reserved, we plated ADSCs in collagen/chitosan scaffolds and compared the proliferation of ADSCs in three dimensional (3D) static environment and 3D dynamic environment. hADSCs were isolated and cultivated in T-flask, in collagen/chitosan scaffolds statically or in the scaffolds placed in spinner flasks for 21 days. During the culture period, the growth dynamic parameters and viability of ADSCs were examined by CCK-8 kit every other day; metabolic rates of glucose and lactic acid were analyzed every day. After 21 days, the cells were evaluated with scanning electron microscope (SEM) for histological examination of the aggregates. The surface markers (CD13, CD29, CD34, CD44, CD45, CD105, CD166 and HLA-DR), the specific transcription factors (Oct-4, Sox-2, Rex-1) and the multi-differentiation potential (adipogenic, osteogenic and chondrogenic) were also assayed to identify the stemness of proliferated cells. The results showed that in spinner flask, the cells in scaffolds could be expanded by more than ten times, and they presented better morphology and vitality and stronger differentiation ability than cells cultivated in scaffolds statically. And the expansion of the both cases for cells in scaffolds was better than that in T-flask. All cells maintained stem cell characteristics after proliferation. From the results, we found that dynamic spinner flask cultivation of hADSC/scaffold constructs resulted in increased proliferation, differentiation and distribution of cells in scaffolds. Therefore, spinner flask cultivation is an easy-to-use inexpensive system for expanding hADSCs in 3D scaffolds.

Three-Dimensional Ingrowth of Bone Cells Within Biodegradable Cryogel Scaffolds in Bioreactors at Different Regimes

Three-dimensional cell ingrowth within biodegradable cryogel scaffolds made of cross-linked 2-hydroxyethyl methacrylate (HEMA)-lactate-dextran with interconnected macropores was studied in bioreactors at different regimes (static, perfusion, and compression-perfusion). An osteoblast-like cell line (MG63) was used in these studies. The samples taken after selected times from the bioreactors were examined by microscopy techniques (light, SEM, TEM, and laser scanning confocal). The cell culture conditions were found to have a significant impact not only on the cell morphology, such as the extent of cell attachment and ingrowth, but also on cellular activities. Dynamic conditions (perfusion and/or compression) greatly improved cell ingrowth and extracellular matrix (ECM) synthesis. Alkaline phosphatase activity results confirmed the positive effect of dynamic conditions on bone cells.

Three-Dimensional Ingrowth of Bone Cells within Biodegradable Cryogel Scaffolds in Bioreactors at Different Regimes

Three-Dimensional Ingrowth of Bone Cells within Biodegradable Cryogel Scaffolds in Bioreactors at Different Regimes

Effects of chondrogenic and osteogenic regulatory factors on composite constructs grown using human mesenchymal stem cells, silk scaffolds and bioreactors

Human mesenchymal stem cells (hMSCs) isolated from bone marrow aspirates were cultured on silk scaffolds in rotating bioreactors for three weeks with either chondrogenic or osteogenic medium supplements to engineer cartilage- or bone-like tissue constructs. Osteochondral composites formed from these cartilage and bone constructs were cultured for an additional three weeks in culture medium that was supplemented with chondrogenic factors, supplemented with osteogenic factors or unsupplemented. Progression of cartilage and bone formation and the integration between the two regions were assessed by medical imaging (magnetic resonance imaging and micro-computerized tomography imaging), and by biochemical, histological and mechanical assays. During composite culture (three to six weeks), bone-like tissue formation progressed in all three media to a markedly larger extent than cartilage-like tissue formation. The integration of the constructs was most enhanced in composites cultured in chondrogenic medium. The results suggest that tissue composites with well-mineralized regions and substantially less developed cartilage regions can be generated in vitro by culturing hMSCs on silk scaffolds in bioreactors, that hMSCs have markedly higher capacity for producing engineered bone than engineered cartilage, and that chondrogenic factors play major roles at early stages of bone formation by hMSCs and in the integration of the two tissue constructs into a tissue composite.