Warren L. Grayson

Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs

Low oxygen tension is thought to be an integral component of the human mesenchymal stem cell (hMSC) native bone marrow microenvironment. HMSC were cultured under physiologically relevant oxygen environments (2% O2) in three‐dimensional (3D) constructs for up to 1 month in order to investigate the combined effects of chronic hypoxia and 3D architecture on hMSC tissue‐development patterns. Hypoxic hMSC exhibited an extended lag phase in order to acclimatize to culture conditions. However, they subsequently proliferated continuously throughout the culture period, while maintaining significantly higher colony‐forming unit capabilities and expressing higher levels of stem cell genes than hMSC cultured at 20% O2 (normoxic) conditions. Upon induction, hypoxic hMSC also expressed higher levels of osteoblastic and adipocytic differentiation markers than normoxic controls. Hypoxia induced increased total protein levels in hMSC throughout the culture period, as well as significantly different fibronectin expression patterns suggesting that oxygen levels can significantly affect tissue‐development patterns. Importantly, hMSC maintained the ability to thrive in prolonged hypoxic conditions suggesting that hypoxia may be an essential element of the in vivo hMSC niche. Further studies are required to determine how variations in cellular characteristics and ECM expression impact on the physiological properties of the engineered tissue, yet these results strongly indicate that oxygen tension is a key parameter that influences the in vitro characteristics of hMSC and their development into tissues. J. Cell. Physiol. 207: 331–339, 2006.

Engineering anatomically shaped human bone grafts

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.

Tissue engineered bone grafts: biological requirements, tissue culture and clinical relevance.

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.

Nucleation and Growth of Mineralized Bone Matrix on Silk-Hydroxyapatite Composite Scaffolds

We describe a composite hydroxyapatite (HA)-silk fibroin scaffold designed to induce and support the formation of mineralized bone matrix by human mesenchymal stem cells (hMSCs) in the absence of osteogenic growth factors. Porous three-dimensional silk scaffolds were extensively used in our previous work for bone tissue engineering and showed excellent biodegradability and biocompatibility. However, silk is not an osteogenic material and has a compressive stiffness significantly lower than that of native bone. In the present study, we explored the incorporation of silk sponge matrices with HA (bone mineral) micro-particles to generate highly osteogenic composite scaffolds capable of inducing the in vitro formation of tissue-engineered bone. Different amounts of HA were embedded in silk sponges at volume fractions of 0%, 1.6%, 3.1% and 4.6% to enhance the osteoconductive activity and mechanical properties of the scaffolds. The cultivation of hMSCs in the silk/HA composite scaffolds under perfusion conditions resulted in the formation of bone-like structures and an increase in the equilibrium Youngs modulus (up to 4-fold or 8-fold over 5 or 10 weeks of cultivation, respectively) in a manner that correlated with the initial HA content. The enhancement in mechanical properties was associated with the development of the structural connectivity of engineered bone matrix. Collectively, the data suggest two mechanisms by which the incorporated HA enhanced the formation of tissue engineered bone: through osteoconductivity of the material leading to increased bone matrix production, and by providing nucleation sites for new mineral resulting in the connectivity of trabecular-like architecture.

Human Mesenchymal Stem Cells Tissue Development in 3D PET Matrices

Human mesenchymal stem cells (hMSCs) are attractive cell sources for engineered tissue constructs with broad therapeutic potential. Three‐dimensional (3D) hMSC tissue development in nonwoven poly(ethylene terephthalate) (PET) fibrous matrices was investigated. HMSCs were seeded onto 3D PET scaffolds and were cultured for over 1 month. Their proliferation rates were affected by seeding density but remained much lower than those of 2D controls. Compared to 2D surfaces, hMSCs grown in 3D scaffolds secreted and embedded themselves in an extensive ECM network composed of collagen I, collagen IV, fibronectin, and laminin. HMSCs were influenced by the orientation of adjacent PET fibers to organize the ECM proteins into highly aligned fibrils. We observed the increased expressions of α2β1 integrin but a slight decrease in the expression of α5β1 integrin in 3D compared to 2D culture and found that αVβ3 was expressed only in 2D. Paxillin expression was down‐regulated in 3D culture with a concomitant change in its localization patterns. We demonstrated the multi‐lineage potentials of the 3D tissue constructs by differentiating the cells grown in the scaffolds into osteoblasts and adipocytes. Taken together, these results showed that hMSCs grown in 3D scaffolds display tissue development patterns distinct from their 2D counterparts and provide important clues for designing 3D scaffolds for developing tissue engineered constructs.

Stromal cells and stem cells in clinical bone regeneration

Stem-cell-mediated bone repair has been used in clinical trials for the regeneration of large craniomaxillofacial defects, to slow the process of bone degeneration in patients with osteonecrosis of the femoral head and for prophylactic treatment of distal tibial fractures. Successful regenerative outcomes in these investigations have provided a solid foundation for wider use of stromal cells in skeletal repair therapy. However, employing stromal cells to facilitate or enhance bone repair is far from being adopted into clinical practice. Scientific, technical, practical and regulatory obstacles prevent the widespread therapeutic use of stromal cells. Ironically, one of the major challenges lies in the limited understanding of the mechanisms via which transplanted cells mediate regeneration. Animal models have been used to provide insight, but these models largely fail to reproduce the nuances of human diseases and bone defects. Consequently, the development of targeted approaches to optimize cell-mediated outcomes is difficult. In this Review, we highlight the successes and challenges reported in several clinical trials that involved the use of bone-marrow-derived mesenchymal or adipose-tissue-derived stromal cells. We identify several obstacles blocking the mainstream use of stromal cells to enhance skeletal repair and highlight technological innovations or areas in which novel techniques might be particularly fruitful in continuing to advance the field of skeletal regenerative medicine.

Engineering custom-designed osteochondral tissue grafts

Tissue engineering is expected to help us outlive the failure of our organs by enabling the creation of tissue substitutes capable of fully restoring the original tissue function. Degenerative joint disease, which affects one-fifth of the US population and is the countrys leading cause of disability, drives current research of actively growing, functional tissue grafts for joint repair. Toward this goal, living cells are used in conjunction with biomaterial scaffolds (serving as instructive templates for tissue development) and bioreactors (providing environmental control and molecular and physical regulatory signals). In this review, we discuss the requirements for engineering customized, anatomically-shaped, stratified grafts for joint repair and the challenges of designing these grafts to provide immediate functionality (load bearing, structural support) and long-term regeneration (maturation, integration, remodeling).

Effects of oxygen transport on 3-d human mesenchymal stem cell metabolic activity in perfusion and static cultures: experiments and mathematical model.

Human mesenchymal stem cells (hMSCs) have unique potential to develop into functional tissue constructs to replace a wide range of tissues damaged by disease or injury. While recent studies have highlighted the necessity for 3‐D culture systems to facilitate the proper biological, physiological, and developmental processes of the cells, the effects of the physiological environment on the intrinsic tissue development characteristics in the 3‐D scaffolds have not been fully investigated. In this study, experimental results from a 3‐D perfusion bioreactor system and the static culture are combined with a mathematical model to assess the effects of oxygen transport on hMSC metabolism and proliferation in 3‐D constructs grown in static and perfusion conditions. Cells grown in the perfusion culture had order of magnitude higher metabolic rates, and the perfusion culture supports higher cell density at the end of cultivation. The specific oxygen consumption rate for the constructs in the perfusion bioreactor was found to decrease from 0.012 to 0.0017 μmol/106 cells/h as cell density increases, suggesting intrinsic physiological change at high cell density. BrdU staining revealed the noneven spatial distribution of the proliferating cells in the constructs grown under static culture conditions compared to the cells that were grown in the perfusion system. The hypothesis that the constructs in static culture grow under oxygen limitation is supported by higher YL/G in static culture. Modeling results show that the oxygen tension in the static culture is lower than that of the perfusion unit, where the cell density was 4 times higher. The experimental and modeling results show the dependence of cell metabolism and spatial growth patterns on the culture environment and highlight the need to optimize the culture parameters in hMSC tissue engineering

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.

Biomimetic approach to tissue engineering

The overall goal of tissue engineering is to create functional tissue grafts that can regenerate or replace our defective or worn out tissues and organs. Examples of grafts that are now in pre-clinical studies or clinical use include engineered skin, cartilage, bone, blood vessels, skeletal muscle, bladder, trachea, and myocardium. Engineered tissues are also finding applications as platforms for pharmacological and physiological studies in vitro. To fully mobilize the cells biological potential, a new generation of tissue engineering systems is now being developed to more closely recapitulate the native developmental milieu, and mimic the physiologic mechanisms of transport and signaling. We discuss the interactions between regenerative biology and engineering, in the context of (i) creation of functional tissue grafts for regenerative medicine (where biological input is critical), and (ii) studies of stem cells, development and disease (where engineered tissues can serve as advanced 3D models).