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All living beings continue their life by receiving energy and by excreting waste products. In animals, the arteries are the pathways of these transfers to the cells. Angiogenesis, the formation of the arteries by the development of pre-existed parental blood vessels, is a phenomenon that occurs naturally during puberty due to certain physiological processes such as menstruation, wound healing, or the adaptation of athletes' bodies during exercise. Nonetheless, the same life-giving process also occurs frequently in some patients and, conversely, occurs slowly in some physiological problems, such as cancer and diabetes, so inhibiting angiogenesis has been considered to be one of the important strategies to fight these diseases. Accordingly, in tissue engineering and regenerative medicine, the highly controlled process of angiogenesis is very important in tissue repairing. Excessive angiogenesis can promote tumor progression and lack of enough angiogensis can hinder tissue repair. Thereby, both excessive and deficient angiogenesis can be problematic, this review article introduces and describes the types of factors involved in controlling angiogenesis. Considering all of the existing strategies, we will try to lay out the latest knowledge that deals with stimulating/inhibiting the angiogenesis. At the end of the article, owing to the early-reviewed mechanical aspects that overshadow angiogenesis, the strategies of angiogenesis in tissue engineering will be discussed.
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Neovascularização Fisiológica , Engenharia Tecidual , Animais , Humanos , Neovascularização Patológica , Medicina Regenerativa , CicatrizaçãoRESUMO
BACKGROUND: Oral mucosa equivalents (OMEs) have been used as in vitro models (eg, for studies of human oral mucosa biology and pathology, toxicological and pharmacological tests of oral care products), and clinically to treat oral defects. However, the human oral mucosa is a highly vascularized tissue and implantation of large OMEs can fail due to a lack of vascularization. To develop equivalents that better resemble the human oral mucosa and increase the success of implantation to repair large-sized defects, efforts have been made to prevascularize these constructs. PURPOSE: The aim of this narrative review is to provide an overview of the human oral mucosa structure, common approaches for its reconstruction, and the development of OMEs, their prevascularization, and in vitro and clinical potential applications. STUDY SELECTION: Articles on non-prevascularized and prevascularized OMEs were included, since the development and applications of non-prevascularized OMEs are a foundation for the design, fabrication, and optimization of prevascularized OMEs. CONCLUSIONS: Several studies have reported the development and in vitro and clinical applications of OMEs and only a few were found on prevascularized OMEs using different approaches of fabrication and incorporation of endothelial cells, indicating a lack of standardized protocols to obtain these equivalents. However, these studies have shown the feasibility of prevascularizing OMEs and their implantation in animal models resulted in enhanced integration and healing. Vascularization in tissue equivalents is still a challenge, and optimization of cell culture conditions, biomaterials, and fabrication techniques along with clinical studies is required.
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Mucosa Bucal , Engenharia Tecidual , Animais , Materiais Biocompatíveis , Células Endoteliais , Humanos , Neovascularização Fisiológica , Engenharia Tecidual/métodosRESUMO
mRNA therapeutics have increased in popularity, largely due to the transient and fast nature of protein expression and the low risk of off-target effects. This has increased drastically with the remarkable success of mRNA-based vaccines for COVID-19. Despite advances in lipid nanoparticle (LNP)-based delivery, the mechanisms that regulate efficient endocytic trafficking and translation of mRNA remain poorly understood. Although it is widely acknowledged that the extracellular matrix (ECM) regulates uptake and expression of exogenous nano-complexed genetic material, its specific effects on mRNA delivery and expression have not yet been examined. Here, we demonstrate a critical role for matrix stiffness in modulating both mRNA transfection and expression and uncover distinct mechano-regulatory mechanisms for endocytosis of mRNA through RhoA mediated mTOR signaling and cytoskeletal dynamics. Our findings have implications for effective delivery of therapeutic mRNA to targeted tissues that may be differentially affected by tissue and matrix stiffness.
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COVID-19 , Nanopartículas , COVID-19/terapia , Vacinas contra COVID-19 , Humanos , Hidrogéis , Lipídeos/genética , Lipossomos , RNA Mensageiro/genéticaRESUMO
Bone is an active organ that continuously undergoes an orchestrated process of remodeling throughout life. Bone tissue is uniquely capable of adapting to loading, hormonal, and other changes happening in the body, as well as repairing bone that becomes damaged to maintain tissue integrity. On the other hand, diseases such as osteoporosis and metastatic cancers disrupt normal bone homeostasis leading to compromised function. Historically, our ability to investigate processes related to either physiologic or diseased bone tissue has been limited by traditional models that fail to emulate the complexity of native bone. Organ-on-a-chip models are based on technological advances in tissue engineering and microfluidics, enabling the reproduction of key features specific to tissue microenvironments within a microfabricated device. Compared to conventional in-vitro and in-vivo bone models, microfluidic models, and especially organs-on-a-chip platforms, provide more biomimetic tissue culture conditions, with increased predictive power for clinical assays. In this review, we will report microfluidic and organ-on-a-chip technologies designed for understanding the biology of bone as well as bone-related diseases and treatments. Finally, we discuss the limitations of the current models and point toward future directions for microfluidics and organ-on-a-chip technologies in bone research.
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Bone mineralization is a highly specific and dynamic nanoscale process that has been studied extensively from a structural, chemical, and biological standpoint. Bone tissue, therefore, may be defined by the interplay of its intricately mineralized matrix and the cells that regulate its biological function. However, the far majority of engineered bone model systems and bone replacement materials have been unable to replicate this key characteristic of bone tissue; that is, the ability of cells to be gradually and rapidly embedded in a three-dimensional (3D) heavily calcified matrix material. Here we review the characteristics that define the bone matrix from a nanostructural perspective. We then revisit the benefits and challenges of existing model systems and engineered bone replacement materials, and discuss recent efforts to replicate the biological, cellular, mechanical, and materials characteristics of bone tissue on the nano- to microscale. We pay particular attention to a recently proposed method developed by our group, which seeks to replicate key aspects of the entrapment of bone cells within a mineralized matrix with precisions down to the level of individual nano-crystallites, inclusive of the bone vasculature, and osteogenic differentiation process. In summary, this paper discusses existing and emerging evidence pointing towards future developments bridging the gap between the fields of biomineralization, structural biology, stem cells, and tissue engineering, which we believe will hold the key to engineer truly functional bone-like tissue in the laboratory.
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Osso e Ossos/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Hidrogéis/farmacologia , Nanoestruturas/química , Diferenciação Celular/efeitos dos fármacos , Humanos , Osteogênese/efeitos dos fármacos , Engenharia Tecidual/métodosRESUMO
AIMS: The structural organization of collagen from mineralized tissues, such as dentin and bone, has been a topic of debate in the recent literature. Recent reports have presented novel interpretations of the complexity of collagen type I at different hierarchical levels and in different tissues. Here, we investigate the nanostructural organization of demineralized dentin collagen following the digestion of non-collagenous components with a trypsin enzyme. MATERIALS AND METHODS: Dentin specimens were obtained from healthy third-molars, cut into small cubes, and polished down to 1 µm roughness. Samples were then demineralized with 10% citric acid for 2 min. Selected specimens were further treated with a solution containing 1 mg/ml trypsin for 48 hours at 37 °C (pH 7.9-9.0). Both untreated and trypsin digested samples were analyzed using SDS-PAGE, Field Emission Scanning Electron Microscopy (FE-SEM), and nanoindentation, where surface hardness and creep properties were compared before and after treatments. RESULTS: FE-SEM images of demineralized dentin showed the banded morphology of D-periodical collagen type I, which upon enzymatic digestion with trypsin appeared to dissociate longitudinally, consistently unraveling ~20 nm structures (microfibril bundles). Such nanoscale structures, to the best of our knowledge, have not been characterized in dentin previously. Mechanical characterization via nanoindentation showed that the unraveling of such microfibril bundles affected the creep displacement and creep rate of demineralized dentin. CONCLUSION: In summary, our results provide novel evidence of the organization of collagen type I from dentin, which may have important implications for the interaction of dental materials with the organic dentin matrix and the mechanical properties of mineralized tissues.
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Colágeno Tipo I/química , Dentina/química , Matriz Extracelular/química , Dente Molar/química , Matriz Extracelular/ultraestrutura , Humanos , Dente Molar/ultraestruturaRESUMO
Survival of functional tissue constructs of clinically relevant size depends on the formation of an organized and uniformly distributed network of blood vessels and capillaries. The lack of such vasculature leads to spatio-temporal gradients in oxygen, nutrients and accumulation of waste products inside engineered tissue constructs resulting in negative biological events at the core of the scaffold. Unavailability of a well-defined vasculature also results in ineffective integration of scaffolds to the host vasculature upon implantation. Arguably, one of the greatest challenges in engineering clinically relevant bone substitutes, therefore, has been the development of vascularized bone scaffolds. Various approaches ranging from peptide and growth factor functionalized biomaterials to hyper-porous scaffolds have been proposed to address this problem with reasonable success. An emerging alternative to address this challenge has been the fabrication of pre-vascularized scaffolds by taking advantage of biomanufacturing techniques, such as soft- and photo-lithography or 3D bioprinting, and cell-based approaches, where functional capillaries are engineered in cell-laden scaffolds prior to implantation. These strategies seek to engineer pre-vascularized tissues in vitro, allowing for improved anastomosis with the host vasculature upon implantation, while also improving cell viability and tissue development in vitro. This book chapter provides an overview of recent methods to engineer pre-vascularized scaffolds for bone regeneration. We first review the development of functional blood capillaries in bony structures and discuss controlled delivery of growth factors, co-culture systems, and on-chip studies to engineer vascularized cell-laden biomaterials. Lastly, we review recent studies using microfabrication techniques and 3D printing to engineer pre-vascularized scaffolds for bone tissue engineering.
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Regeneração Óssea/fisiologia , Osso e Ossos/fisiologia , Neovascularização Fisiológica/fisiologia , Engenharia Tecidual/métodos , Alicerces Teciduais , Materiais Biocompatíveis/metabolismo , Osso e Ossos/irrigação sanguínea , Osso e Ossos/citologia , Técnicas de Cocultura/métodos , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Engenharia Tecidual/tendênciasRESUMO
Tissue development, function, and disease are largely driven by the spatial organization of individual cells and their cell-cell interactions. Precision engineered tissues with single-cell spatial resolution, therefore, have tremendous potential for next generation disease models, drug discovery, and regenerative therapeutics. Despite significant advancements in biofabrication approaches to improve feature resolution, strategies to fabricate tissues with the exact same organization of individual cells in their native cellular microenvironment have remained virtually non-existent to date. Here we report a method to spatially pattern single cells with up to eight cell phenotypes and subcellular spatial precision. As proof-of-concept we first demonstrate the ability to systematically assess the influence of cellular microenvironments on cell behavior by controllably altering the spatial arrangement of cell types in bioprinted precision cell-cell interaction arrays. We then demonstrate, for the first time, the ability to produce high-fidelity replicas of a patient's annotated cancer biopsy with subcellular resolution. The ability to replicate native cellular microenvironments marks a significant advancement for precision biofabricated in-vitro models, where heterogenous tissues can be engineered with single-cell spatial precision to advance our understanding of complex biological systems in a controlled and systematic manner.
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Bone defects may occur in different sizes and shapes due to trauma, infections, and cancer resection. Autografts are still considered the primary treatment choice for bone regeneration. However, they are hard to source and often create donor-site morbidity. Injectable microgels have attracted much attention in tissue engineering and regenerative medicine due to their ability to replace inert implants with a minimally invasive delivery. Here, we developed novel cell-laden bioprinted gelatin methacrylate (GelMA) injectable microgels, with controllable shapes and sizes that can be controllably mineralized on the nanoscale, while stimulating the response of cells embedded within the matrix. The injectable microgels were mineralized using a calcium and phosphate-rich medium that resulted in nanoscale crystalline hydroxyapatite deposition and increased stiffness within the crosslinked matrix of bioprinted GelMA microparticles. Next, we studied the effect of mineralization in osteocytes, a key bone homeostasis regulator. Viability stains showed that osteocytes were maintained at 98 % viability after mineralization with elevated expression of sclerostin in mineralized compared to non-mineralized microgels, showing that mineralization can effectively enhances osteocyte maturation. Based on our findings, bioprinted mineralized GelMA microgels appear to be an efficient material to approximate the bone microarchitecture and composition with desirable control of sample injectability and polymerization. These bone-like bioprinted mineralized biomaterials are exciting platforms for potential minimally invasive translational methods in bone regenerative therapies.
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Gelatina , Microgéis , Gelatina/farmacologia , Gelatina/química , Materiais Biocompatíveis , Metacrilatos/químicaRESUMO
Bone autografts remain the gold standard for bone grafting surgeries despite having increased donor site morbidity and limited availability. Bone morphogenetic protein-loaded grafts represent another successful commercial alternative. However, the therapeutic use of recombinant growth factors has been associated with significant adverse clinical outcomes. This highlights the need to develop biomaterials that closely approximate the structure and composition of bone autografts, which are inherently osteoinductive and biologically active with embedded living cells, without the need for added supplements. Here, injectable growth factor-free bone-like tissue constructs are developed, that closely approximate the cellular, structural, and chemical composition of bone autografts. It is demonstrated that these micro-constructs are inherently osteogenic, and demonstrate the ability to stimulate mineralized tissue formation and regenerate bone in critical-sized defects in-vivo. Furthermore, the mechanisms that allow human mesenchymal stem cells (hMSCs) to be highly osteogenic in these constructs, despite the lack of osteoinductive supplements, are assessed, whereby Yes activated protein (YAP) nuclear localization and adenosine signaling appear to regulate osteogenic cell differentiation. The findings represent a step toward a new class of minimally invasive, injectable, and inherently osteoinductive scaffolds, which are regenerative by virtue of their ability to mimic the tissue cellular and extracellular microenvironment, thus showing promise for clinical applications in regenerative engineering.
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Microgéis , Humanos , Regeneração Óssea/fisiologia , Osteogênese/fisiologia , Osso e Ossos , Materiais Biocompatíveis/química , Diferenciação Celular/fisiologia , Engenharia Tecidual , Alicerces Teciduais/químicaRESUMO
Successful integration of cell-laden tissue constructs with host vasculature depends on the presence of functional capillaries to provide oxygen and nutrients to the embedded cells. However, diffusion limitations of cell-laden biomaterials challenge regeneration of large tissue defects that require bulk-delivery of hydrogels and cells. Herein, a strategy to bioprint geometrically controlled, endothelial and stem-cell laden microgels in high-throughput is introduced, allowing these cells to form mature and functional pericyte-supported vascular capillaries in vitro, and then injecting these pre-vascularized constructs minimally invasively in-vivo. It is demonstrated that this approach offers both desired scalability for translational applications as well as unprecedented levels of control over multiple microgel parameters to design spatially-tailored microenvironments for better scaffold functionality and vasculature formation. As a proof-of-concept, the regenerative capacity of the bioprinted pre-vascularized microgels is compared with that of cell-laden monolithic hydrogels of the same cellular and matrix composition in hard-to-heal defects in vivo. The results demonstrate that the bioprinted microgels have faster and higher connective tissue formation, more vessels per area, and widespread presence of functional chimeric (human and murine) vascular capillaries across regenerated sites. The proposed strategy, therefore, addresses a significant issue in regenerative medicine, demonstrating a superior potential to facilitate translational regenerative efforts.
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Bioimpressão , Microgéis , Camundongos , Humanos , Animais , Engenharia Tecidual/métodos , Bioimpressão/métodos , Materiais Biocompatíveis , Hidrogéis , Alicerces Teciduais , Impressão TridimensionalRESUMO
Augmenting adaptive immunity is a critical goal for developing next-generation cancer therapies. T and B cells infiltrating the tumor dramatically influence cancer progression through complex interactions with the local microenvironment. Cancer cells evade and limit these immune responses by hijacking normal immunologic pathways. Current experimental models using conventional primary cells, cell lines, or animals have limitations for studying cancer-immune interactions directly relevant to human biology and clinical translation. Therefore, engineering methods to emulate such interplay at local and systemic levels are crucial to expedite the development of better therapies and diagnostic tools. In this review, we discuss the challenges, recent advances, and future directions toward engineering the tumor-immune microenvironment (TME), including key elements of adaptive immunity. We first offer an overview of the recent research that has advanced our understanding of the role of the adaptive immune system in the tumor microenvironment. Next, we discuss recent developments in 3D in-vitro models and engineering approaches that have been used to study the interaction of cancer and stromal cells with B and T lymphocytes. We summarize recent advancement in 3D bioengineering and discuss the need for 3D tumor models that better incorporate elements of the complex interplay of adaptive immunity and the tumor microenvironment. Finally, we provide a perspective on current challenges and future directions for modeling cancer-immune interactions aimed at identifying new biological targets for diagnostics and therapeutics.
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Neoplasias , Animais , Humanos , Neoplasias/patologia , Microambiente TumoralRESUMO
Bone defects may occur in different sizes and shapes due to trauma, infections, and cancer resection. Autografts are still considered the primary treatment choice for bone regeneration. However, they are hard to source and often create donor-site morbidity. Injectable microgels have attracted much attention in tissue engineering and regenerative medicine due to their ability to replace inert implants with a minimally invasive delivery. Here, we developed novel cell-laden bioprinted gelatin methacrylate (GelMA) injectable microgels, with controllable shapes and sizes that can be controllably mineralized on the nanoscale, while stimulating the response of cells embedded within the matrix. The injectable microgels were mineralized using a calcium and phosphate-rich medium that resulted in nanoscale crystalline hydroxyapatite deposition and increased stiffness within the crosslinked matrix of bioprinted GelMA microparticles. Next, we studied the effect of mineralization in osteocytes, a key bone homeostasis regulator. Viability stains showed that osteocytes were maintained at 98% viability after mineralization with elevated expression of sclerostin in mineralized compared to non-mineralized microgels, indicating that mineralization effectively enhances osteocyte maturation. Based on our findings, bioprinted mineralized GelMA microgels appear to be an efficient material to approximate the bone microarchitecture and composition with desirable control of sample injectability and polymerization. These bone-like bioprinted mineralized biomaterials are exciting platforms for potential minimally invasive translational methods in bone regenerative therapies.
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Bioprinting has emerged as one of the most promising strategies for fabrication of functional organs in the lab as an alternative to transplant organs. While progress in the field has mostly been restricted to a few miniaturized tissues with minimal biological functionality until a few years ago, recent progress has advanced the concept of building three-dimensional multicellular organ complexity remarkably. This review discusses a series of milestones that have paved the way for bioprinting of tissue constructs that have advanced levels of biological and architectural functionality. Critical materials, engineering and biological challenges that are key to addressing the desirable function of engineered organs are presented. These are discussed in light of the many difficulties to replicate the heterotypic organization of multicellular solid organs, the nanoscale precision of the extracellular microenvironment in hierarchical tissues, as well as the advantages and limitations of existing bioprinting methods to adequately overcome these barriers. In summary, the advances of the field toward realistic manufacturing of functional organs have never been so extensive, and this manuscript serves as a road map for some of the recent progress and the challenges ahead.
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Bioimpressão , Bioimpressão/métodos , Impressão Tridimensional , Engenharia Tecidual/métodosRESUMO
Dental caries is a biofilm-mediated, diet-modulated, multifactorial and dynamic disease that affects more than 90% of adults in Western countries. The current treatment for decayed tissue is based on using materials to replace the lost enamel or dentin. More than 500 million dental restorations are placed annually worldwide, and materials used for these purposes either directly or indirectly interact with dentin and pulp tissues. The development and understanding of the effects of restorative dental materials are based on different in-vitro and in-vivo tests, which have been evolving with time. In this review, we first discuss the characteristics of the tooth and the dentin-pulp interface that are unique for materials testing. Subsequently, we discuss frequently used in-vitro tests to evaluate the biocompatibility of dental materials commonly used for restorative procedures. Finally, we present our perspective on the future directions for biological research on dental materials using tissue engineering and organs on-a-chip approaches. STATEMENT OF SIGNIFICANCE: Dental caries is still the most prevalent infectious disease globally, requiring more than 500 million restorations to be placed every year. Regrettably, the failure rates of such restorations are still high. Those rates are partially based on the fact that current platforms to test dental materials are somewhat inaccurate in reproducing critical components of the complex oral microenvironment. Thus, there is a collective effort to develop new materials while evolving the platforms to test them. In this context, the present review critically discusses in-vitro models used to evaluate the biocompatibility of restorative dental materials and brings a perspective on future directions for tissue-engineered and organs-on-a-chip platforms for testing new dental materials.
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Cárie Dentária , Dentina , Adulto , Resinas Compostas , Materiais Dentários/farmacologia , Restauração Dentária Permanente , Humanos , Dispositivos Lab-On-A-Chip , Teste de MateriaisRESUMO
It has long been proposed that recapitulating the extracellular matrix (ECM) of native human tissues in the laboratory may enhance the regenerative capacity of engineered scaffoldsin-vivo. Organ- and tissue-derived decellularized ECM biomaterials have been widely used for tissue repair, especially due to their intrinsic biochemical cues that can facilitate repair and regeneration. The main purpose of this study was to synthesize a new photocrosslinkable human bone-derived ECM hydrogel for bioprinting of vascularized scaffolds. To that end, we demineralized and decellularized human bone fragments to obtain a bone matrix, which was further processed and functionalized with methacrylate groups to form a photocrosslinkable methacrylate bone ECM hydrogel- bone-derived biomaterial (BoneMA). The mechanical properties of BoneMA were tunable, with the elastic modulus increasing as a function of photocrosslinking time, while still retaining the nanoscale features of the polymer networks. The intrinsic cell-compatibility of the bone matrix ensured the synthesis of a highly cytocompatible hydrogel. The bioprinted BoneMA scaffolds supported vascularization of endothelial cells and within a day led to the formation of interconnected vascular networks. We propose that such a quick vascular network formation was due to the host of pro-angiogenic biomolecules present in the bone ECM matrix. Further, we also demonstrate the bioprintability of BoneMA in microdimensions as injectable ECM-based building blocks for microscale tissue engineering in a minimally invasive manner. We conclude that BoneMA may be a useful hydrogel system for tissue engineering and regenerative medicine.
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Bioimpressão , Bioimpressão/métodos , Células Endoteliais , Matriz Extracelular/química , Humanos , Hidrogéis/química , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
INTRODUCTION: The transplantation of stem cells/tissue constructs into root canal space is a promising strategy for regenerating lost pulp tissue. However, the root canal system, which is cone shaped with a taper from the larger coronal end to the smaller apical end, limits the vascular supply and, therefore, the regenerative capacity. The current study aimed to fabricate built-in microchannels with different tapers to explore various approaches to endothelialize these microchannels. METHODS: The fluidic microchannels with varying tapers (parallel, 0.04, and 0.06) were fabricated within gelatin methacryloyl (GelMA) hydrogel (with or without stem cell from the apical papilla [SCAP] encapsulation) of different concentrations (5%, 7.5%, and 10% [w/v]). Green fluorescent protein-expressing human umbilical vein endothelial cells (HUVECs-GFP) were seeded alone or with SCAPs in coculture into these microchannels. Angiogenic sprouting was assessed by fluorescence and a confocal microscope and ImageJ software (National Institutes of Health, Bethesda, MD). Immunostaining was conducted to illustrate monolayer formation. Data were statistically analyzed by 1-way/2-way analysis of variance. RESULTS: HUVEC-only inoculation formed an endothelial monolayer inside the microchannel without angiogenic sprouting. HUVECs-GFP/SCAPs cocultured at a 1:1 ratio produced the longest sprouting compared with the other 3 ratios. The average length of the sprouting in the 0.04 taper microchannel was significantly longer compared with that in the parallel and 0.06 taper microchannels. Significant differences in HUVEC-GFP sprouting were observed in 5% GelMA hydrogel. Encapsulation of SCAPs within hydrogel further stimulated the sprouting of HUVECs. CONCLUSIONS: The coculture of SCAPs and HUVECs-GFP at a ratio of 1:1 in 0.04 taper fluidic microchannels fabricated with 5% (w/v) GelMA hydrogel with SCAPs encapsulated was found to be the optimal condition to enhance angiogenesis inside tapered microchannels.
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Gelatina , Hidrogéis , Polpa Dentária , Células Endoteliais da Veia Umbilical Humana , Humanos , Neovascularização FisiológicaRESUMO
Conventional biomaterials developed for bone regeneration fail to fully recapitulate the nanoscale structural organization and complex composition of the native bone microenvironment. Therefore, despite promoting osteogenic differentiation of stem cells, they fall short of providing the structural, biochemical, and mechanical stimuli necessary to drive osteogenesis for bone regeneration and function. To address this, we have recently developed a novel strategy to engineer bone-like tissue using a biomimetic approach to achieve rapid and controlled nanoscale mineralization of a cell-laden matrix in the presence of osteopontin, a non-collagenous protein, and a supersaturated solution of calcium and phosphate medium. Here, we build on this approach to engineer bone regeneration scaffolds comprising methacrylated gelatin (GelMA) hydrogels incorporated with calcium citrate core-shell microparticles as a sustained and reliable source of calcium ions for in situ mineralization. We demonstrate successful biomineralization of GelMA hydrogels by embedded calcium carbonate-calcium citrate core-shell microparticles with the resultant mineral chemistry, structure, and organization reminiscent of that of native bone. The biomimetic mineralization was further shown to promote osteogenic differentiation of encapsulated human mesenchymal stem cells even in the absence of other exogenous osteogenic induction factors. Ultimately, by combining the superior biological response engendered by biomimetic mineralization with the intrinsic tissue engineering advantages offered by GelMA, such as biocompatibility, biodegradability, and printability, we envision that our system offers great potential for bone regeneration efforts.
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Gelatina/química , Hidrogéis/química , Células-Tronco Mesenquimais/fisiologia , Metacrilatos/química , Carbonato de Cálcio , Citrato de Cálcio , Diferenciação Celular , Sobrevivência Celular , Humanos , Osteogênese , Tamanho da PartículaRESUMO
Adequate vascularization of scaffolds is a prerequisite for successful repair and regeneration of lost and damaged tissues. It has been suggested that the maturity of engineered vascular capillaries, which is largely determined by the presence of functional perivascular mural cells (or pericytes), plays a vital role in maintaining vessel integrity during tissue repair and regeneration. Here, we investigated the role of pericyte-supported-engineered capillaries in regenerating bone in a critical-size rat calvarial defect model. Prior to implantation, human umbilical vein endothelial cells and human bone marrow stromal cells (hBMSCs) were cocultured in a collagen hydrogel to induce endothelial cell morphogenesis into microcapillaries and hBMSC differentiation into pericytes. Upon implantation into the calvarial bone defects (8 mm), the prevascularized hydrogels showed better bone formation than either untreated controls or defects treated with autologous bone grafts (positive control). Bone formation parameters such as bone volume, coverage area, and vascularity were significantly better in the prevascularized hydrogel group than in the autologous bone group. Our results demonstrate that tissue constructs engineered with pericyte-supported vascular capillaries may approximate the regenerative capacity of autologous bone, despite the absence of osteoinductive or vasculogenic growth factors.
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Células Imobilizadas , Hidrogéis , Células-Tronco Mesenquimais , Crânio , Animais , Células Imobilizadas/metabolismo , Células Imobilizadas/patologia , Células Imobilizadas/transplante , Xenoenxertos , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Ratos , Ratos Nus , Crânio/irrigação sanguínea , Crânio/lesões , Crânio/metabolismo , Crânio/patologiaRESUMO
Successful bone healing in severe trauma depends on early revascularization to restore oxygen, nutrient, growth factor, and progenitor cell supply to the injury. Therapeutic angiogenesis strategies have therefore been investigated to promote revascularization following severe bone injuries; however, results have been inconsistent. This is the first study investigating the effects of dual angiogenic growth factors (VEGF and PDGF) with low-dose bone morphogenetic protein-2 (BMP-2; 2.5 µg) on bone healing in a clinically challenging composite bone-muscle injury model. Our hydrogel-based delivery systems demonstrated a more than 90% protein entrapment efficiency and a controlled simultaneous release of three growth factors over 28 days. Co-stimulation of microvascular fragment constructs with VEGF and PDGF promoted vascular network formation in vitro compared to VEGF or PDGF alone. In an in vivo model of segmental bone and volumetric muscle loss injury, combined VEGF (5 µg) and PDGF (7.5 µg or 15 µg) delivery with a low dose of BMP-2 significantly enhanced regeneration of vascularized bone compared to BMP-2 treatment alone. Notably, the regenerated bone mechanics reached ~60% of intact bone, a value that was previously only achieved by delivery of high-dose BMP-2 (10 µg) in this injury model. Overall, sustained delivery of VEGF, PDFG, and BMP-2 is a promising strategy to promote functional vascularized bone tissue regeneration following severe composite musculoskeletal injury. Although this study is conducted in a clinically relevant composite injury model in rats using a simultaneous release strategy, future studies are necessary to test the regenerative potential of spatiotemporally controlled delivery of triple growth factors on bone healing using large animal models. STATEMENT OF SIGNIFICANCE: Volumetric muscle loss combined with delayed union or non-union bone defect causes deleterious effects on bone regeneration even with the supplementation of bone morphogenetic protein-2 (BMP-2). In this study, the controlled delivery of dual angiogenic growth factors (vascular endothelial growth factor [VEGF] + Platelet-derived growth factor [PDGF]) increases vascular growth in vitro. Co-delivering VEGF+PDGF significantly increase the bone formation efficacy of low-dose BMP-2 and improves the mechanics of regenerated bone in a challenging composite bone-muscle injury model.