The ability to engineer anatomically correct pieces of viable and functional human bone would have tremendous potential for bone reconstructions after congenital defects, cancer resections, and trauma. We report that clinically sized, anatomically shaped, viable human bone grafts can be engineered by using human mesenchymal stem cells (hMSCs) and a "biomimetic" scaffold-bioreactor system. We selected the temporomandibular joint (TMJ) condylar bone as our tissue model, because of its clinical importance and the challenges associated with its complex shape. Anatomically shaped scaffolds were generated from fully decellularized trabecular bone by using digitized clinical images, seeded with hMSCs, and cultured with interstitial flow of culture medium. A bioreactor with a chamber in the exact shape of a human TMJ was designed for controllable perfusion throughout the engineered construct. By 5 weeks of cultivation, tissue growth was evidenced by the formation of confluent layers of lamellar bone (by scanning electron microscopy), markedly increased volume of mineralized matrix (by quantitative microcomputer tomography), and the formation of osteoids (histologically). Within bone grafts of this size and complexity cells were fully viable at a physiologic density, likely an important factor of graft function. Moreover, the density and architecture of bone matrix correlated with the intensity and pattern of the interstitial flow, as determined in experimental and modeling studies. This approach has potential to overcome a critical hurdle-in vitro cultivation of viable bone grafts of complex geometries-to provide patient-specific bone grafts for craniofacial and orthopedic reconstructions.biomimetic | bioreactor | craniofacial regeneration | mesenchymal stem cells | temporomandibular joint | tissue engineering

The tremendous need for bone tissue in numerous clinical situations and the limited availability of suitable bone grafts are driving the development of tissue engineering approaches to bone repair. In order to engineer viable bone grafts, one needs to understand the mechanisms of native bone development and fracture healing, as these processes should ideally guide the selection of optimal conditions for tissue culture and implantation. Engineered bone grafts have been shown to have capacity for osteogenesis, osteoconduction, osteoinduction and osteointegration - functional connection between the host bone and the graft. Cells from various anatomical sources in conjunction with scaffolds and osteogenic factors have been shown to form bone tissue in vitro. The use of bioreactor systems to culture cells on scaffolds before implantation further improved the quality of the resulting bone grafts. Animal studies confirmed the capability of engineered grafts to form bone and integrate with the host tissues. However, the vascularization of bone remains one of the hurdles that need to be overcome if clinically sized, fully viable bone grafts are to be engineered and implanted. We discuss here the biological guidelines for tissue engineering of bone, the bioreactor cultivation of human mesenchymal stem cells on three-dimensional scaffolds, and the need for vascularization and functional integration of bone grafts following implantation.

Stem cells have the ability for prolonged self-renewal and differentiation into mature cells of various lineages, which makes them important cell sources for tissue engineering applications. Their remarkable ability to replenish and differentiate in vivo is regulated by both intrinsic and extrinsic cellular mechanisms. The anatomical location where the stem cells reside, known as the "stem cell niche or microenvironment," provides signals conducive to the maintenance of definitive stem cell properties. Physiological condition including oxygen tension is an important component of the stem cell microenvironment and has been shown to play a role in regulating both embryonic and adult stem cells. This review focuses on oxygen as a signaling molecule and the way it regulates the stem cells' development into mesenchymal tissues in vitro. The physiological relevance of low oxygen tension as an environmental parameter that uniquely benefits stem cells' expansion and maintenance is described along with recent findings on the regulatory effects of oxygen on embryonic stem cells and adult mesenchymal stem cells. The relevance to tissue engineering is discussed in the context of the need to specifically regulate the oxygen content in the cellular microenvironment in order to optimize in vitro tissue development.

We report engineering of half centimeter size bone constructs created in vitro using human Adipose-derived Stem Cells (hASC), decellularized bone scaffolds and perfusion bioreactors. The hASCs are easily accessible, can be used in an autologous fashion, are rapidly expanded in culture, and are capable of osteogenic differentiation. hASCs from four donors were characterized for their osteogenic capacity, and one representative cell population was used for tissue engineering experiments. Culture-expanded hASCs were seeded on fully decellularized native bone scaffolds (4 mm Ø × 4 mm) providing the necessary structural and mechanical environment for osteogenic differentiation, and cultured in bioreactors with medium perfusion. The interstitial flow velocity was set to a level necessary to maintain cell viability and function throughout the construct volume (400 μs−1), via enhanced mass transport. After 5 weeks of cultivation, the addition of osteogenic supplements (dexamethasone, sodium-β-glycerophosphate, ascorbic acid-2-phosphate) to culture medium significantly increased the construct cellularity and the amounts of bone matrix components (collagen, bone sialoprotein, bone osteopontin). Medium perfusion markedly improved the distribution of cells and bone matrix in engineered constructs. In summary, a combination of hASCs, decellularized bone scaffold, perfusion culture and osteogenic supplements resulted in the formation of compact and viable bone tissue constructs.

There is a critical need to increase the size of bone grafts that can be cultured in vitro for use in regenerative medicine. Perfusion bioreactors have been used to improve the nutrient and gas transfer capabilities and reduce the size limitations inherent to static culture, as well as to modulate cellular responses by hydrodynamic shear. Our aim was to understand the effects of medium flow velocity on cellular phenotype and the formation of bone-like tissues in three-dimensional engineered constructs. We utilized custom-designed perfusion bioreactors to culture bone constructs for five weeks using a wide range of superficial flow velocities (80, 400, 800, 1200 and 1800 μm/s), corresponding to estimated initial shear stresses ranging from 0.6 – 20 mPa. Increasing the flow velocity significantly affected cell morphology, cell-cell interactions, matrix production and composition, and the expression of osteogenic genes. Within the range studied, the flow velocities ranging from 400 to 800 μm/s yielded the best overall osteogenic responses. Using mathematical models, we determined that even at the lowest flow-velocity (80 μm/s) the oxygen provided was sufficient to maintain viability of the cells within the construct. Yet it was clear that this flow-velocity did not adequately support the development of bone-like tissue. The complexity of the cellular responses found at different flow-velocities underscores the need to use a range of evaluation parameters to determine the quality of engineered-bone.

These data demonstrate the clinical potential of covalently bound VAN and CAS on Ti to reduce microbial biofilm formation without jeopardizing osseointegration.

Fully bio-based 3-dimensional porous scaffolds based on freeze-dried cellulose nanofibers (70-90 wt%) stabilized using a genipin crosslinked matrix of gelatin and chitosan were prepared. Morphology studies using scanning electron microscopy showed that the scaffolds have interconnected pores with average pore diameters of 75-200 mm and nanoscaled pore wall roughness, both favorable for cell interactions with cartilage repair. X-ray tomography confirmed the 3-dimensional homogeneity and interconnectivity of the pores as well as the fibrillar structure of the scaffolds. The compression modulus of the scaffolds (1-3 MPa) at room conditions was higher than natural cartilage (z1 MPa). The lowered compression modulus of 10-60 kPa in phosphate buffered saline (PBS) at 37 C was considered favorable for chondrogenesis. The current study therefore successfully addressed the challenge of tailoring the pore structure and mechanical properties simultaneously for cartilage regeneration. Furthermore, the scaffolds' high porosity (z95%), high PBS uptake and good cytocompatibility towards chondrocytes are considered beneficial for cell attachment and extracellular matrix (ECM) production.

This article discusses parameters relevant for successful translation of research on different cell sources into clinically applicable cell therapies: the influence of the intervertebral disc microenvironment on the cell phenotype, issues associated with cell culture and technical preparation of cell products, as well as discussing current regulatory requirements. There are advantages and disadvantages of each proposed cell type, but no strong evidence to favour any one particular cell source at the moment.