Context Of Tissue Engineering Research Articles

Role of tendon-derived stem cells in tendon and ligament repair: focus on tissue engineer.

This review offered a comprehensive analysis of tendon and ligament injuries, emphasizing the crucial role of tendon-derived stem cells (TDSCs) in tissue engineering as a potential solution for these challenging medical conditions. Tendon and ligament injuries, prevalent among athletes, the elderly, and laborers, often result in long-term disability and reduced quality of life due to the poor intrinsic healing capacity of these avascular structures. The formation of biomechanically inferior scar tissue and a high rate of reinjury underscore the need for innovative approaches to enhance and guide the regenerative process. This review delved into the complexities of tendon and ligament structure and function, types of injuries and their impacts, and the limitations of the natural repair process. It particularly focused on the role of TDSCs within the context of tissue engineering. TDSCs, with their ability to differentiate into tenocytes, are explored in various applications, including biocompatible scaffolds for cell tracking, co-culture systems to optimize tendon-bone healing, and graft healing techniques. The review also addressed the challenges of immunoreactivity post-transplantation, the importance of pre-treating TDSCs, and the potential of hydrogels and decellularized matrices in supporting tendon regeneration. It concluded by highlighting the essential roles of mechanical and molecular stimuli in TDSC differentiation and the current challenges in the field, paving the way for future research directions.

Papillary and reticular fibroblasts generate distinct microenvironments that differentially impact angiogenesis.

Papillary and reticular dermis show distinct extracellular matrix (ECM) and vascularization corresponding to their specific functions. These characteristics are associated with gene expression patterns of fibroblasts freshly isolated from their native microenvironment. In order to assess the relevance of these fibroblast subpopulations in a tissue engineering context, we investigated their contribution to matrix production and vascularization using cell sheet culture conditions. We first performed RNA-seq differential expression analysis to determine whether several rounds of cell amplification and high-density culture affected their gene expression profile. Bioinformatics analysis revealed that expression of angiogenesis-related and matrisome gene signatures were maintained, resulting in papillary and reticular ECMs that differ in composition and structure. The impact of secreted or ECM-associated factors was then assessed using two independent 3D angiogenesis assays: -1/ a fibrin hydrogel-based assay allowing investigation of diffusible secreted factors, -2/ a scaffold-free cell-sheet based assay for investigation of fibroblast-produced microenvironment. These analyses revealed that papillary fibroblasts secrete highly angiogenic factors and produce a microenvironment characterised by ECM remodelling capacity and dense and branched microvascular network, whereas reticular fibroblasts produced more structural core components of the ECM associated with less branched and larger vessels. These features mimick the characteristics of both the ECM and the vasculature of dermis subcompartments. In addition to showing that skin fibroblast populations differentially regulate angiogenesis via both secreted and ECM factors, our work emphasizes the importance of papillary and reticular fibroblasts for engineering and modelling dermis microenvironment and vascularization. STATEMENT OF SIGNIFICANCE: Recent advances have brought to the forefront the central role of microenvironment and vascularization in tissue engineering for regenerative medicine and microtissue modelling. We have investigated the role of papillary and reticular fibroblast subpopulations using scaffold-free cell sheet culture. This approach provides differentiated cells conditions allowing the production of their own microenvironment. Analysis of gene expression profiles and characterisation of the matrix produced revealed strong and specific angiogenic properties that we functionally characterized using 3D angiogenesis models targeting the respective role of either secreted or matrix-bound factors. This study demonstrates the importance of cell-generated extracellular matrix and questions the importance of cell source and the relevance of hydrogels for developing physio-pathologically relevant tissue engineered substitutes.

Hydrogels for 3D bioprinting in tissue engineering and regenerative medicine: Current progress and challenges.

Three-dimensional (3D) bioprinting is a promising and innovative biomanufacturing technology, which can achieve precise position controlling of cells and extracellular matrix components, and further create complex and functional multi-cellular tissues or organs in a 3D environment. Bioink in the form of the cell-loaded hydrogel is most commonly used in bioprinting, and it is vital to the process of bioprinting. The bionic scaffold should possess suitable mechanical strength, biocompatibility, cell proliferation, survival, and other biological characteristics. The disadvantages of natural polymer hydrogel materials include poor mechanical properties as well as low printing performance and shape fidelity. Over the past years, a series of synthetic, modified, and nanocomposite hydrogels have been developed, which can interact through physical interactions, chemical covalent bond crosslinking, and bioconjugation reactions to change the characteristics to satisfy the requirements. In this review, a comprehensive summary is provided on recent research regarding the unique properties of hydrogel bioinks for bioprinting, with optimized methods and technologies highlighted, which have both high-value research significance and potential clinical applications. A critical analysis of the strengths and weaknesses of each hydrogel-based biomaterial ink is presented at the beginning or end of each section, alongside the latest improvement strategies employed by current researchers to address their respective shortcomings. Furthermore, we propose potential repair sites for each hydrogel-based ink based on their distinctive repair features, while reflecting on current research limitations. Finally, we synthesize and analyze expert opinions on the future of these hydrogel-based bioinks in the broader context of tissue engineering and regenerative medicine, offering valuable insights for future investigations.

Tissues with Patterned Vessels or Protein Release Induce Vascular Chemotaxis in an In Vitro Platform.

Engineered tissues designed for translational applications in regenerative medicine require vascular networks to deliver oxygen and nutrients rapidly to the implanted cells. A limiting factor of in vivo translation is the rapid and successful inosculation, or connection, of host and implanted vascular networks and subsequent perfusion of the implant. An approach gaining favor in vascular tissue engineering is to provide instructive cues from the engineered tissue to enhance host vascular penetration and connection with the implant. Here, we use a novel in vitro platform based on the aortic ring assay to evaluate the impact of patterned, endothelialized vessels or growth factor release from engineered constructs on preinosculative vascular cell outgrowth from surrogate host tissue in a controlled, defined environment, and introduce robust tools for evaluating vascular morphogenesis and chemotaxis. We demonstrate the creation of engineered vessels at the arteriole scale, which develop basement membrane, exhibit tight junctions, and actively sprout into the surrounding bulk hydrogel. Vessel-containing constructs are co-cultured adjacent to rodent aortic rings, and the resulting heterocellular outgrowth is quantified. Cells originating from the aortic ring migrate preferentially toward constructs containing engineered vessels with 1.5-fold faster outgrowth kinetics, 2.5-fold increased cellular density, and 1.6-fold greater network formation versus control (no endothelial cells and growth factor-reduced culture medium). Growth factor release from constructs with nonendothelialized channels and in reduced factor medium equivalently stimulates sustained vascular outgrowth distance, cellular density, and network formation, akin to engineered vessels in endothelial growth medium 2 (EGM-2) medium. In conclusion, we show that three-dimensional endothelialized patterned vessels or growth factor release stimulate a robust, host-derived vascular cell chemotactic response at early time points critical for instructive angiogenic cues. Further, we developed robust, unbiased tools to quantify metrics of vascular morphogenesis and preinosculative heterocellular outgrowth from rat aortic rings and demonstrated the utility of our complex, controlled environment, heterocellular in vitro platform. Impact statement Using a novel in vitro platform, we show that engineered constructs with patterned vessels or angiogenic growth factor release, two methods of instructing host revascularization responses, equivalently improve early host-derived vascular outgrowth. Our platform leverages the aortic ring assay in a tissue engineering context to study preinosculative vascular cell chemotaxis from surrogate host vascular cells in response to paracrine cues from co-cultured engineered tissues using robust, open-source quantification tools. Our accessible and flexible platform enables translationally focused studies in revascularization using implantable therapeutics containing prepatterned vessels with greater environmental control than in vivo studies to advance vascular tissue engineering.

MicroCT analysis of connectivity in porous structures: optimizing data acquisition and analytical methods in the context of tissue engineering.

Micro-computed X-ray tomography (MicroCT) is one of the most powerful techniques available for the three-dimensional characterization of complex multi-phase or porous microarchitectures. The imaging and analysis of porous networks are of particular interest in tissue engineering due to the ability to predict various large-scale cellular phenomena through the micro-scale characterization of the structure. However, optimizing the parameters for MicroCT data capture and analyses requires a careful balance of feature resolution and computational constraints while ensuring that a structurally representative section is imaged and analysed. In this work, artificial datasets were used to evaluate the validity of current analytical methods by considering the effect of noise and pixel size arising from the data capture, and intrinsic structural anisotropy and heterogeneity. A novel ‘segmented percolation method’ was developed to exclude the effect of anomalous, non-representative features within the datasets, allowing for scale-invariant structural parameters to be obtained consistently and without manual intervention for the first time. Finally, an in-depth assessment of the imaging and analytical procedures are presented by considering percolation events such as micro-particle filtration and cell sieving within the context of tissue engineering. Along with the novel guidelines established for general pixel size selection for MicroCT, we also report our determination of 3 μm as the definitive pixel size for use in analysing connectivity for tissue engineering applications.

Dynamic Bioreactors with Integrated Microfabricated Devices for Mechanobiological Screening.

Biomechanical stimulation is a common strategy to improve the growth, maturation, and function of a variety of engineered tissues. However, identifying optimized biomechanical conditioning protocols is challenging, as cell responses to mechanical stimuli are modulated by other multifactorial microenvironmental cues, including soluble factors and biomaterial properties. Traditional bioreactors lack the throughput necessary for combinatorial testing of cell activity in mechanically stimulated engineered tissues. Microfabricated systems can improve experimental throughput, but often do not provide uniform mechanical loading, are challenging to use, lack robustness, and offer limited amounts of cells and tissue for analysis. To address the need for higher-throughput, combinatorial testing of cell activity in a tissue engineering context, we developed a hybrid approach, in which flexible polydimethylsiloxane microfabricated inserts were designed to simultaneously generate multiple tensile strains when stretched cyclically in a standard dynamic bioreactor. In the embodiment presented in this study, each insert contained an array of 35 dog bone-shaped wells in which cell-seeded microscale hydrogels can be polymerized, with up to eight inserts stretched simultaneously in the bioreactor. Uniformity of the applied strains, both along the length of a microtissue and across multiple microtissues at the same strain level, was confirmed experimentally. In proof-of-principle experiments, the combinatorial effects of dynamic strain, biomaterial stiffness, and transforming growth factor (TGF)-β1 stimulation on myofibroblast differentiation were tested, revealing both known and novel interaction effects and suggesting tissue engineering strategies to regulate myofibroblast activation. This platform is expected to have wide applicability in systematically probing combinations of mechanobiological tissue engineering parameters for desired effects on cell fate and tissue function. Impact Statement In this study, we introduce a dynamic bioreactor system incorporating microfabricated inserts to enable systematic probing of the effects of combinations of mechanobiological parameters on engineered tissues. This novel platform offers the ease of use, robustness, and well-defined mechanical strain stimuli inherent in traditional dynamic bioreactors, but significantly improves throughput (up to 280 microtissues can be tested simultaneously in the embodiment presented in this study). This platform has wide applicability to systematically probe combinations of dynamic mechanical strain, biomaterial properties, biochemical stimulation, and other parameters for desired effects on cell fate and engineered tissue development.

Modelling multi-scale cell–tissue interaction of tissue-engineered muscle constructs

Expectation on engineered tissue substitute continues to grow, and for an effective development of a functional tissue and to control its quality, cellular mechanoresponse plays a key role. Although the mechanoresponse – in terms of cell–tissue interaction across scales – has been understood better in recent years, there are still technical limitations to quantitatively monitor the processes involved in the development of both native and engineered tissues. Computational (in silico) studies have been utilised to complement the experimental limitations and successfully applied to the prediction of tissue growth. We here review recent activities in the area of combined experimental and computational analyses of tissue growth, especially in the tissue engineering context, and highlight the advantages of such an approach for the future of the tissue engineering, using our own case study of predicting musculoskeletal tissue engineering construct development.

Biomimetic microenvironment complexity to redress the balance between biodegradation and de novo matrix synthesis during early phase of vascular tissue engineering

Physiological functionality of a tissue engineered vascular construct depends on the phenotype of smooth muscle cells (SMCs) cultured into the scaffold and mechanical robust of the construct relies on two simultaneous mechanisms including scaffold biodegradation and de novo matrix synthesis by SMCs which both can be influenced by scaffold properties and culture condition. Our focus in this study was to provide an appropriate environmental condition within tissue engineering context to meet foregoing requisites for a successful vascular regeneration. To this end, SMCs seeded onto electrospun Tecophilic/gelatin (TP(70)/gel(30)) scaffolds were subjected to orbital shear stress. Given the improvement in mechanical properties of dynamically stimulated cell-seeded constructs after a span of 10days, effect of fluctuating shear stress on scaffold biodegradation and SMC behavior was investigated. Compared to static condition, SMCs proliferated more rapidly and concomitantly built up greater collagen content in response to dynamic culture, suggesting a reasonable balance between scaffold biodegradation and matrix turnover for maintaining the structural integrity and mechanical support to seeded cells during early phase of vascular tissue engineering. Despite higher proliferation of SMCs under dynamic condition, cells preserved nearly spindle like morphology and contractile protein expression likely thanks to composition of the scaffold.

Fabrication of Biodegradable Synthetic Vascular Networks and Their Use as a Model of Angiogenesis

One of the greatest challenges currently faced in tissue engineering is the incorporation of vascular networks within tissue-engineered constructs. The aim of this study was to develop a technique for producing a perfusable, 3-dimensional, cell-friendly model of vascular structures that could be used to study the factors affecting angiogenesis and vascular biology in engineered systems in more detail. Initially, biodegradable synthetic pseudovascular networks were produced via the combination of robocasting and electrospinning techniques. The internal surfaces of the vascular channels were then recellularized with human dermal microvascular endothelial cells (HDMECs) with and without the presence of human dermal fibroblasts (HDFs) on the outer surface of the scaffold. After 7 days in culture, channels that had been reseeded with HDMECs alone demonstrated irregular cell coverage. However, when using a co-culture of HDMECs inside and HDFs outside the vascular channels, coverage was found to be continuous throughout the internal channel. Using this cell combination, collagen gels loaded with vascular endothelial growth factor were deposited onto the outer surface of the scaffold and cultured for a further 7 days. After this, endothelial cell outgrowth from within the channels into the collagen gel was observed, showing that the engineered vasculature maintains its capacity for angiogenesis. Furthermore, the HDMECs appeared to have formed perfusable tubules within the gel. These results show promising steps towards the development of an in vitro platform for studying angiogenesis and vascular biology in a tissue engineering context.

Immunomodulatory polymeric scaffold enhances extracellular matrix production in cell co-cultures under dynamic mechanical stimulation

Despite the importance of immune cells in regulating the wound healing process following injury, there are few examples of synthetic biomaterials that have the capacity to push the body’s immune cells toward pro-regeneration phenotypes, and fewer still that are designed with the intention of achieving this immunomodulatory character. While monocytes and their derived macrophages have been recognized as important contributors to tissue remodeling in vivo, this is primarily believed to be due to their ability to regulate other cell types. The ability of monocytes and macrophages to generate tissue products themselves, however, is currently not well appreciated within the field of tissue regeneration. Furthermore, while monocytes/macrophages are found in remodeling tissue that is subjected to mechanical loading, the effect this biomechanical strain on monocytes/macrophages and their ability to regulate tissue-specific cellular activity has not been understood due to the complexity of the many factors involved in the in vivo setting, hence necessitating the use of controlled in vitro culture platforms to investigate this phenomenon. In this study, human monocytes were co-cultured with human coronary artery smooth muscle cells (VSMCs) on a tubular (3mm ID) degradable polyurethane scaffold, with a unique combination of non-ionic polar, hydrophobic and ionic chemistry (D-PHI). The goal was to determine if such a synthetic matrix could be used in a co-culture system along with dynamic biomechanical stimulus (10% circumferential strain, 1Hz) conditions in order to direct monocytes to enhance tissue generation, and to better comprehend the different ways in which monocytes/macrophages may contribute to new tissue production. Mechanical strain and monocyte co-culture had a complementary and non-mitigating effect on VSMC growth. Co-culture samples demonstrated increased deposition of sulphated glycosaminoglycans (GAGs) and elastin, as well as increases in the release of FGF-2, a growth factor that can stimulate VSMC growth, while dynamic culture supported increases in collagen I and III as well as increased mechanical properties (elastic modulus, tensile strength) vs. static controls. Macrophage polarization toward an M1 state was not promoted by the biomaterial or culture conditions tested. Monocytes/macrophages cultured on D-PHI were also shown to produce vascular extracellular matrix components, including collagen I, collagen III, elastin, and GAGs. This study highlights the use of synthetic biomaterials having immunomodulatory character in order to promote cell and tissue growth when used in tissue engineering strategies, and identifies ECM deposition by monocytes/macrophages as an unexpected source of this new tissue. Statement of SignificanceThe ability of biomaterials to regulate macrophage activation towards a wound healing phenotype has recently been shown to support positive tissue regeneration. However, the ability of immunomodulatory biomaterials to harness monocyte/macrophage activity to support tissue engineering strategies in vitro holds enormous potential that has yet to be investigated. This study used a monocyte co-culture on a degradable polyurethane (D-PHI) to regulate the response of VSMCs in combination with biomechanical strain in a vascular tissue engineering context. Results demonstrate that immunomodulatory biomaterials, such as D-PHI, that support a desirable macrophage activation state can be combined with biomechanical strain to augment vascular tissue production in vitro, in part due to the novel and unexpected contribution of monocytes/macrophages themselves producing vascular ECM proteins.

Pre-crosslinked polymeric collagen in 3-D models of mechanically stiff tissues: Blended collagen polymer hydrogels for rapid layer fabrication

Currently one factor hindering the development of collagen hydrogel constructs for tissue engineering is the mismatch between initial cellularity and mechanical strength. The main advantage of collagen hydrogel tissue constructs is their ability to support interstitially seeded cells. However, cells are sensitive to their environment, in particular, substrate stiffness, which cannot easily be replicated within hydrogels without cytotoxic cross-linking treatment. In this study, pre-crosslinked polymeric collagen fibrils are introduced as a starting material, thereby avoiding artificial cross-linking. Shear aggregation of this material in solution results in fibril alignment, but cell addition is only possible when polymeric collagen is blended with its monomeric counterparts to slow the aggregation of collagen fibrils. The hydrogel can then be brought to physiological collagen density by plastic compression. Interstitially seeded fibroblasts were supported for 14days. Although compression of blended gels resulted in some cell death due to increased rate of fluid expulsion, not normally seen in conventional collagen hydrogels, the surviving cell population recovers during subsequent culture. Importantly, the compression process can be controlled and customized to limit cell damage. This is the first report of native polymeric collagen used in a tissue engineering context, for the rapid production of a stiff collagen-cell constructs.

Macrophage-mediated angiogenic activation of outgrowth endothelial cells in co-culture with primary osteoblasts

The successful vascularisation of complex tissue engineered constructs for bone regeneration is still a major challenge in the field of tissue engineering. In this context, co-culture systems of endothelial cells and osteoblasts represent a promising approach to advance the formation of a stable vasculature as well as an excellent in vitro model to identify factors that positively influence bone healing processes, including angiogenesis. Under physiological conditions, the activation phase of angiogenesis is mainly induced by hypoxia or inflammation. Inflammatory cells such as macrophages secrete proinflammatory cytokines and proangiogenic growth factors, finally leading to the formation of new blood vessels. The aim of this study was to investigate if macrophages might positively influence the formation of microvessel-like structures via inflammatory mechanisms in a co-culture system consisting of human outgrowth endothelial cells (OECs) and primary osteoblasts. Treatment of co-cultures with macrophages (induced from THP-1) resulted in a higher number of microvessel-like structures formed by OECs compared to the co-culture. This change correlated with a significantly higher concentration of the proangiogenic VEGF in cell culture supernatants of triple-cultures and was accompanied by an increase in the expression of different proinflammatory cytokines, such as IL-6, IL-8 and TNFα. In addition, the expression of E-selectin and ICAM-1, adhesion molecules which are strongly involved in the interaction between leukocytes and endothelial cells during the process of inflammation was also found to be higher in triple-cultures compared to the double co-cultures, documenting an ongoing proinflammatory stimulus. These results raise the possibility of actively using pro-inflammatory stimuli in a tissue engineering context to accelerate healing mechanisms.

Cell microenvironment engineering and monitoring for tissue engineering and regenerative medicine: the recent advances.

In tissue engineering and regenerative medicine, the conditions in the immediate vicinity of the cells have a direct effect on cells' behaviour and subsequently on clinical outcomes. Physical, chemical, and biological control of cell microenvironment are of crucial importance for the ability to direct and control cell behaviour in 3-dimensional tissue engineering scaffolds spatially and temporally. In this review, we will focus on the different aspects of cell microenvironment such as surface micro-, nanotopography, extracellular matrix composition and distribution, controlled release of soluble factors, and mechanical stress/strain conditions and how these aspects and their interactions can be used to achieve a higher degree of control over cellular activities. The effect of these parameters on the cellular behaviour within tissue engineering context is discussed and how these parameters are used to develop engineered tissues is elaborated. Also, recent techniques developed for the monitoring of the cell microenvironment in vitro and in vivo are reviewed, together with recent tissue engineering applications where the control of cell microenvironment has been exploited. Cell microenvironment engineering and monitoring are crucial parts of tissue engineering efforts and systems which utilize different components of the cell microenvironment simultaneously can provide more functional engineered tissues in the near future.

Efficient in vivo vascularization of tissue-engineering scaffolds

The success of tissue engineering depends on the rapid and efficient formation of a functional blood vasculature. Adult blood vessels comprise endothelial cells and perivascular mural cells that assemble into patent tubules ensheathed by a basement membrane during angiogenesis. Using individual vessel components, we characterized intra-scaffold microvessel self-assembly efficiency in a physiological in vivo tissue engineering implant context. Primary human microvascular endothelial and vascular smooth muscle cells were seeded at different ratios in poly-L-lactic acid (PLLA) scaffolds enriched with basement membrane proteins (Matrigel) and implanted subcutaneously into immunocompromised mice. Temporal intra-scaffold microvessel formation, anastomosis and perfusion were monitored by immunohistochemical, flow cytometric and in vivo multiphoton fluorescence microscopy analysis. Vascularization in the tissue-engineering context was strongly enhanced in implants seeded with a complete complement of blood vessel components: human microvascular endothelial and vascular smooth muscle cells in vivo assembled a patent microvasculature within Matrigel-enriched PLLA scaffolds that anastomosed with the host circulation during the first week of implantation. Multiphoton fluorescence angiographic analysis of the intra-scaffold microcirculation showed a uniform, branched microvascular network. 3D image reconstruction analysis of human pulmonary artery smooth muscle cell (hPASMC) distribution within vascularized implants was non-random and displayed a preferential perivascular localization. Hence, efficient microvessel self-assembly, anastomosis and establishment of a functional microvasculture in the native hypoxic in vivo tissue engineering context is promoted by providing a complete set of vascular components.

In Vitro and In Vivo Bioluminescent Imaging of Hypoxia in Tissue-Engineered Grafts

Survival and growth of cellular grafts in tissue engineering (TE) are limited by the rate of oxygen (O(2)) and nutrient diffusion. As such, monitoring the levels of nutrients and O(2) available to the cells is essential to assess the physiology of the cells and to evaluate strategies aiming at improving nutrient availability. In this article, a reporter system containing the luciferase gene driven by a hypoxia responsive promoter was used to monitor cellular hypoxia in a TE context. We report that luciferase activity correlates with the O(2) tension in the cell culture medium. When transgenic cells were seeded onto scaffolds and implanted in immune-deficient mice subcutaneously, luciferase activity was detected. To validate the response to O(2) levels of this reporter system, we cultured transgenic cells on biomaterials in a flow perfusion bioreactor and observed that cells in the bioreactor displayed a drastically lower luciferase activity than conventional static culture, and that higher luciferase activity is observed in the interior of a tissue-engineered construct, illustrating the uneven O(2) distribution in three-dimensional constructs under conventional static culture. We conclude that this reporter system is a versatile tool to investigate cellular O(2) availability in TE both in vitro and in vivo.

Engineered Microenvironments for Controlled Stem Cell Differentiation

In a developing organism, tissues emerge from coordinated sequences of cell renewal, differentiation, and assembly that are orchestrated by spatial and temporal gradients of multiple regulatory factors. The composition, architecture, signaling, and biomechanics of the cellular microenvironment act in concert to provide the necessary cues regulating cell function in the developing and adult organism. With recent major advances in stem cell biology, tissue engineering is becoming increasingly oriented toward biologically inspired in vitro cellular microenvironments designed to guide stem cell growth, differentiation, and functional assembly. The premise is that to unlock the full potential of stem cells, at least some aspects of the dynamic three-dimensional (3D) environments that are associated with their renewal, differentiation, and assembly in native tissues need to be reconstructed. In the general context of tissue engineering, we discuss the environments for guiding stem cell function by an interactive use of biomaterial scaffolds and bioreactors, and focus on the interplay between molecular and physical regulatory factors. We highlight some illustrative examples of controllable cell environments developed through the interaction of stem cell biology and tissue engineering at multiple levels.

Physiology of onlay bone healing

Onlay bone grafting represents one strategy for locally increasing bone stock. This may be due to the need to restore bone in a large defect site such as the use of either autologous bone, or other products, in cranial reconstruction; or the need to provide load-bearing bone for the placement of implants as witnessed in “J” grafts and wedge grafts in the alveolar bone. Clearly, the basic phenomena of host bone healing, comprising the response to surgical intervention, will be common to all cases. However, where inlay grafts are surrounded by host bone tissue, onlay grafts are placed onto the bone surface where the recipient bed may be poorly vascularized cortical bone with a paucity of osteogenic precursors to initiate new bone matrix formation. For this reason, various strategies are available to enhance the wound healing dynamics of onlay grafts. These include the addition of autogenous (platelet products) and/or purified (growth factors) stimulants to healing, the use of membranes in an attempt to control the cellular composition at the wound site, or the augmentation of the host bone with artificial materials. The latter, in the tissue-engineering context, may be combined with both cells and/or growth factors to optimize healing. The long-term response to the graft will be dependent upon the type of graft employed and, importantly, on the biomechanical constraints of the anatomical placement site. Thus, an onlay graft employed in the reconstruction of the zygoma will need to provide long-term mechanical tissue support while on onlay graft in the maxillary sinus may function almost as an inlay graft which can become completely remodeled in time to only that bone which supports an endosseous implant.