PhD Candidate in Bone Tissue Engineering, Eindhoven University of Technology
How to make mini-bones in the laboratory that help us to understand bone development and diseases
Bone is a dynamic tissue that is constantly broken down and built up to the point where every 7 years we essentially have a new bone. It can adapt its mass, structure and geometry according to changing external stimuli. There are 3 main cell types responsible for this remodeling process: osteoblasts, osteoclasts and osteocytes. The osteoblasts are the bone forming cells that build the extracellular protein matrix where mineralization takes place. The osteoclasts do exactly the opposite: they dissolve the crystalized minerals and matrix. The tight regulation of these two opposing tasks is crucial for overall bone health which brings us to the osteocyte. Osteocytes start out as osteoblasts, but they become trapped in the extracellular matrix that they have built. Osteocytes develop long finger-like processes that protrude through the surrounding matrix and connect to other cells. They transform into sensing cells that pick up, send out and transmit signals such as mechanical stimuli to neighboring cells. All the special features of bone are derived from interactions between these three different cell types.
To produce bone in the laboratory, 3D scaffolds are used where stem cells can attach to and turn into osteoblasts and osteocytes in an almost real-life setting. We use different growth factors, chemical compounds and bioreactors that pump fluid through the center of the scaffold and provide the mechanical stimuli that the cells need to produce bone.
These mini-bones allow us to understand how bone develops, how cells interact with each other and how they respond to changes. Unraveling this complex communication network will not only enhance our fundamental knowledge of biological processes in bone but also help us understanding bone related diseases such as brittle bone disease, osteoporosis or bone cancer.
Abstract: Treatment of extensive bone defects requires autologous bone grafting or implantation of bone substitute materials. An attractive alternative has been to engineer fully viable, biological bone grafts in vitro by culturing osteogenic cells within three-dimensional scaffolds, under conditions supporting bone formation. Such grafts could be used for implantation, but also as physiologically relevant models in basic and translational studies of bone development, disease and drug discovery. A source of human cells that can be derived in large numbers from a small initial harvest and predictably differentiated into bone forming cells is critically important for engineering human bone grafts. We discuss the characteristics and limitations of various types of human embryonic and adult stem cells, and their utility for bone tissue engineering.
Pub.: 20 Jul '10, Pinned: 17 Oct '17
Abstract: One of the goals of bone tissue engineering is to design delivery methods for skeletal stem/progenitor cells to repair or replace bone. Although the materials used to retain cells play a central role in the quality of the constructs, the source of cells is key for bone regeneration. Bone marrow is the most common cell source, but other tissues are now being explored, such as the periosteum, fat, muscle, cord blood, and embryonic or induced pluripotent stem cells. The therapeutic effect of exogenous stem/progenitor cells is accepted, yet their contribution to bone repair is not well defined. The in vitro osteo- and/or chondrogenic potential of these skeletal progenitors do not necessarily predict their differentiation potential in vivo and their function may be affected by their ability to home correctly to bone. This review provides an overview of animal models used to test the efficacy of cell-based approaches. We examine the mechanisms of endogenous cell recruitment during bone repair and compare the role of local versus systemic cell recruitment. We discuss how the normal repair process can help define efficacious cell sources for bone tissue engineering and improve their methods of delivery.
Pub.: 10 Sep '11, Pinned: 14 Oct '17
Abstract: The role of Bone Tissue Engineering in the field of Regenerative Medicine has been the topic of substantial research over the past two decades. Technological advances have improved orthopaedic implants and surgical techniques for bone reconstruction. However, improvements in surgical techniques to reconstruct bone have been limited by the paucity of autologous materials available and donor site morbidity. Recent advances in the development of biomaterials have provided attractive alternatives to bone grafting expanding the surgical options for restoring the form and function of injured bone. Specifically, novel bioactive (second generation) biomaterials have been developed that are characterised by controlled action and reaction to the host tissue environment, whilst exhibiting controlled chemical breakdown and resorption with an ultimate replacement by regenerating tissue. Future generations of biomaterials (third generation) are designed to be not only osteoconductive but also osteoinductive, i.e. to stimulate regeneration of host tissues by combining tissue engineering and in situ tissue regeneration methods with a focus on novel applications. These techniques will lead to novel possibilities for tissue regeneration and repair. At present, tissue engineered constructs that may find future use as bone grafts for complex skeletal defects, whether from post-traumatic, degenerative, neoplastic or congenital/developmental "origin" require osseous reconstruction to ensure structural and functional integrity. Engineering functional bone using combinations of cells, scaffolds and bioactive factors is a promising strategy and a particular feature for future development in the area of hybrid materials which are able to exhibit suitable biomimetic and mechanical properties. This review will discuss the state of the art in this field and what we can expect from future generations of bone regeneration concepts.
Pub.: 01 Sep '13, Pinned: 14 Oct '17
Abstract: Natural bone consists of cortical and trabecular morphologies, the latter having variable pore sizes. This study aims at engineering different bone-like structures using scaffolds with small pores (112-224 microm) in diameter on one side and large pores (400-500 microm) on the other, while keeping scaffold porosities constant among groups. We hypothesized that tissue engineered bone-like structure resulting from silk fibroin (SF) implants is pre-determined by the scaffolds' geometry. To test this hypothesis, SF scaffolds with different pore diameters were prepared and seeded with human mesenchymal stem cells (hMSC). As compared to static seeding, dynamic cell seeding in spinner flasks resulted in equal cell viability and proliferation, and better cell distribution throughout the scaffold as visualized by histology and confocal microscopy, and was, therefore, selected for subsequent differentiation studies. Differentiation of hMSC in osteogenic cell culture medium in spinner flasks for 3 and 5 weeks resulted in increased alkaline phosphatase activity and calcium deposition when compared to control medium. Micro-computed tomography (microCT) detailed the pore structures of the newly formed tissue and suggested that the structure of tissue-engineered bone was controlled by the underlying scaffold geometry.
Pub.: 10 Nov '06, Pinned: 17 Oct '17
Abstract: The formation of bone-like tissue from human mesenchymal stem cells (hMSC) cultured in osteogenic medium on silk fibroin scaffolds was monitored and quantified over 44 days in culture using non-invasive time-lapsed micro-computed tomography (microCT). Each construct was imaged nine times in situ. From microCT imaging, detailed morphometrical data on bone volume density, surface-to-volume ratio, trabecular thickness, trabecular spacing, and the structure model index and tissue mineral density were obtained. microCT irradiation did not impact the osteogenic performance of hMSCs based on DNA content, alkaline phosphatase activity, and calcium deposition when compared to non-exposed control samples. Bone-like tissue formation initiated at day 10 of the culture with the deposition of small mineralized clusters. Tissue mineral density increased linearly over time. The surface-to-volume ratio of the bone-like tissues converged asymptotically to 26 mm(-1). Although in vitro formation of bone-like tissue started from clusters, the overall bone volume was not predictable from the time, number, and size of initially formed bone-like clusters. Based on microstructural analysis, the morphometry of the tissue-engineered constructs was found to be in the range of human trabecular bone. In future studies, non-invasive, time-lapsed monitoring may enable researchers to culture tissues in vitro, right until the development of a desired morphology is accomplished. Our data demonstrate the feasibility of qualitatively and quantitatively detailing the spatial and temporal mineralization of bone-like tissue formation in tissue engineering.
Pub.: 05 Jun '07, Pinned: 17 Oct '17
Abstract: Bone (re)-generation and bone fixation strategies utilize biomaterial implants, which are gradually replaced by autologous tissues. Ideally, these biomaterials should be biodegradable, osteoconductive, and provide mechanical strength and integrity until newly formed host tissues can maintain function. Some protein-based biomaterials such as collagens are promising because of their biological similarities to natural proteins on bone surfaces. However, their use as bone implant materials is largely hampered by poor mechanical properties. In contrast, silks offer distinguishing mechanical properties that are tailorable, along with slow degradability to permit adequate time for remodeling. To assess the suitability of silk-based biomaterials as implants for bone healing, we explored the use of novel porous silk fibroin scaffolds as templates for the engineering of bone tissues starting from human bone marrow derived stem cells cultured under osteogenic conditions for up to 5 weeks. The slowly degrading protein matrix permitted adequate temporal control of hydroxyapatite deposition and resulted in the formation of a trabecular-like bone matrix in bioreactor studies. The organic and inorganic components of the engineered bone tissues resembled those of bone, as shown by gene expression analysis, biochemical assays, and X-ray diffractometry. Implantation of the tissue-engineered bone implants (grown in bioreactors for 5 weeks prior to implantation) into calvarial critical size defects in mice demonstrated the capacity of these systems to induce advanced bone formation within 5 weeks, whereas the implantation of stem cell loaded silk scaffolds, and scaffolds alone resulted in less bone formation. These results demonstrate the feasibility of silk-based implants with engineered bone for the (re-)generation of bone tissues and expand the class of protein-based bone-implant materials with a mechanically stable and durable option.
Pub.: 06 Sep '05, Pinned: 17 Oct '17
Abstract: Tissue engineering has emerged as a possible alternative to current treatments for bone injuries and defects. However, the common tissue engineering approach presents some obstacles to the development of functional tissues, such as insufficient nutrient and metabolite transport and non-homogenous cell distribution. Culture of bone cells in three-dimensional constructs in bioreactor systems is a solution for those problems as it improves mass transport in the culture system. For bone tissue engineering spinner flasks, rotating wall vessels and perfusion systems have been investigated, and based on these, variations that support cell seeding and mechanical stimulation have also been researched. This review aims at providing an overview of the concepts, advantages and future applications of bioreactor systems for bone tissue engineering with emphasis on the design of different perfusion systems and parameters that can be optimized.
Pub.: 20 Mar '13, Pinned: 14 Oct '17
Abstract: The purpose of this review is to provide a brief overview of bioreactor-based culture systems as alternatives to conventional two- and three- dimensional counterparts. The role, challenges and future aspirations of bioreactors in the musculoskeletal field (e.g. cartilage, intervertebral disc, tendon and bone) are discussed. Bioreactors, by recapitulating physiological processes, can be used effectively as part of the initial in vitro screening, reducing that way the number of animal required for preclinical assessment, complying with the 3R principles and, in most cases, allowing working with human tissues. The clinical significance of bioreactors is that, by providing more physiologically-relevant conditions to customarily used two- and three- dimensional cultures, they hold the potential to provide a testing platform that is more predictable of a whole tissue response, thereby facilitating the screening of treatments before the initiation of clinical trials. This article is protected by copyright. All rights reserved.
Pub.: 19 Jul '17, Pinned: 15 Oct '17
Abstract: We describe a novel bioreactor system for tissue engineering of bone that enables cultivation of up to six tissue constructs simultaneously, with direct perfusion and imaging capability. The bioreactor was used to investigate the relative effects of initial seeding density and medium perfusion rate on the growth and osteogenic differentiation patterns of bone marrow-derived human mesenchymal stem cells (hMSCs) cultured on three-dimensional scaffolds. Fully decellularized bovine trabecular bone was used as a scaffold because it provided suitable "biomimetic" topography, biochemical composition, and mechanical properties for osteogenic differentiation of hMSCs. Trabecular bone plugs were completely denuded of cellular material using a serial treatment with hypotonic buffers and detergents, seeded with hMSCs, and cultured for 5 weeks. Increasing seeding density from 30 x 10(6) cells/mL to 60 x 10(6) cells/mL did not measurably influence the characteristics of tissue-engineered bone, in contrast to an increase in the perfusion rate from 100 microms(-1) to 400 microms(-1), which radically improved final cell numbers, cell distributions throughout the constructs, and the amounts of bone proteins and minerals. Taken together, these findings suggest that the rate of medium perfusion during cultivation has a significant effect on the characteristics of engineered bone.
Pub.: 16 Jul '08, Pinned: 17 Oct '17
Abstract: 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 5 weeks using a wide range of superficial flow velocities (80, 400, 800, 1,200, and 1,800 µm/s), corresponding to estimated initial shear stresses ranging from 0.6 to 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.
Pub.: 31 Mar '11, Pinned: 14 Oct '17
Abstract: Osteocytes comprise the overwhelming majority of cells in bone and are its only true "permanent" resident cell population. In recent years, conceptual and technological advances on many fronts have helped to clarify the role osteocytes play in skeletal metabolism and the mechanisms they use to perform them. The osteocyte is now recognized as a major orchestrator of skeletal activity, capable of sensing and integrating mechanical and chemical signals from their environment to regulate both bone formation and resorption. Recent studies have established that the mechanisms osteocytes use to sense stimuli and regulate effector cells (e.g., osteoblasts and osteoclasts) are directly coupled to the environment they inhabit-entombed within the mineralized matrix of bone and connected to each other in multicellular networks. Communication within these networks is both direct (via cell-cell contacts at gap junctions) and indirect (via paracrine signaling by secreted signals). Moreover, the movement of paracrine signals is dependent on the movement of both solutes and fluid through the space immediately surrounding the osteocytes (i.e., the lacunar-canalicular system). Finally, recent studies have also shown that the regulatory capabilities of osteocytes extend beyond bone to include a role in the endocrine control of systemic phosphate metabolism. This review will discuss how a highly productive combination of experimental and theoretical approaches has managed to unearth these unique features of osteocytes and bring to light novel insights into the regulatory mechanisms operating in bone.
Pub.: 18 Sep '13, Pinned: 14 Oct '17
Abstract: Osteocytes, the cells residing within the bone matrix and comprising 90% to 95% of the all bone cells, have long been considered quiescent bystander cells compared to the osteoblasts and osteoclasts whose activities cause bone gain and loss, and whose dysfunction lead to growth defects and osteoporosis. However, recent studies show that osteocytes play a crucial, central role in regulating the dynamic nature of bone in all its diverse functions. Osteocytes are now known to be the principal sensors for mechanical loading of bone. They produce the soluble factors that regulate the onset of both bone formation and resorption. Osteocytes regulate local mineral deposition and chemistry at the bone matrix level, and they also function as endocrine cells producing factors that target distant organs such as the kidney to regulate phosphate transport. Osteocytes appear to be the major local orchestrator of many of bone's functions.
Pub.: 04 May '12, Pinned: 14 Oct '17
Abstract: Osteocytes, the most abundant cell population of the bone lineage, have been a major focus in the bone research field in recent years. This population of cells that resides within mineralized matrix is now thought to be the mechanosensory cell in bone and plays major roles in the regulation of bone formation and resorption. Studies of osteocytes had been impaired by their location, resulting in numerous attempts to isolate primary osteocytes and to generate cell lines representative of the osteocytic phenotype. Progress has been achieved in recent years by utilizing in vivo genetic technology and generation of osteocyte directed transgenic and gene deficiency mouse models. We will provide an overview of the current in vitro and in vivo models utilized to study osteocyte biology. We discuss generation of osteocyte-like cell lines and isolation of primary osteocytes and summarize studies that have utilized these cellular models to understand the functional role of osteocytes. Approaches that attempt to selectively identify and isolate osteocytes using fluorescent protein reporters driven by regulatory elements of genes that are highly expressed in osteocytes will be discussed. In addition, recent in vivo studies utilizing overexpression or conditional deletion of various genes using dentin matrix protein (Dmp1) directed Cre recombinase are outlined. In conclusion, evaluation of the benefits and deficiencies of currently used cell lines/genetic models in understanding osteocyte biology underlines the current progress in this field. The future efforts will be directed towards developing novel in vitro and in vivo models that would additionally facilitate in understanding the multiple roles of osteocytes.
Pub.: 18 Oct '12, Pinned: 17 Oct '17
Abstract: Mechanical loading plays a major role in bone remodeling and fracture healing. Mimicking the concept of mechanical loading of bone has been widely studied in bone tissue engineering by perfusion cultures. Nevertheless, there is still debate regarding the in-vitro mechanical stimulation regime. This study aims at investigating the effect of two different flow rates (vlow = 0.001m/s and vhigh = 0.061m/s) on the growth of mineralized tissue produced by human mesenchymal stromal cells cultured on 3-D silk fibroin scaffolds. The flow rates applied were chosen to mimic the mechanical environment during early fracture healing or during bone remodeling, respectively. Scaffolds cultured under static conditions served as a control. Time-lapsed micro-computed tomography showed that mineralized extracellular matrix formation was completely inhibited at vlow compared to vhigh and the static group. Biochemical assays and histology confirmed these results and showed enhanced osteogenic differentiation at vhigh whereas the amount of DNA was increased at vlow. The biological response at vlow might correspond to the early stage of fracture healing, where cell proliferation and matrix production is prominent. Visual mapping of shear stresses, simulated by computational fluid dynamics, to 3-D micro-computed tomography data revealed that shear stresses up to 0.39mPa induced a higher DNA amount and shear stresses between 0.55mPa and 24mPa induced osteogenic differentiation. This study demonstrates the feasibility to drive cell behavior of human mesenchymal stromal cells by the flow velocity applied in agreement with mechanical loading mimicking early fracture healing (vlow) or bone remodeling (vhigh). These results can be used in the future to tightly control the behavior of human mesenchymal stromal cells towards proliferation or differentiation. Additionally, the combination of experiment and simulation presented is a strong tool to link biological responses to mechanical stimulation and can be applied to various in-vitro cultures to improve the understanding of the cause-effect relationship of mechanical loading.
Pub.: 08 Jul '17, Pinned: 14 Oct '17
Abstract: Perfusion culture of mesenchymal stem cells (MSCs) seeded in biomaterial scaffolds provides nutrients for cell survival, enhances extracellular matrix deposition, and increases osteogenic cell differentiation. However, there is no consensus on the appropriate perfusion duration of cellular constructs in vitro to boost their bone forming capacity in vivo. We investigated this phenomenon by culturing human MSCs in macroporous composite scaffolds in a direct perfusion bioreactor and compared their response to scaffolds in continuous dynamic culture conditions on an XYZ shaker. Cell seeding in continuous perfusion bioreactors resulted in more uniform MSC distribution than static seeding. We observed similar calcium deposition in all composite scaffolds over 21 days of bioreactor culture, regardless of pore size. Compared to scaffolds in dynamic culture, perfused scaffolds exhibited increased DNA content and expression of osteogenic markers up to 14 days in culture that plateaued thereafter. We then evaluated the effect of perfusion culture duration on bone formation when MSC-seeded scaffolds were implanted in a murine ectopic site. Human MSCs persisted in all scaffolds at 2 weeks in vivo, and we observed increased neovascularization in constructs cultured under perfusion for 7 days relative to those cultured for 1 day within each gender. At 8 weeks post-implantation, we observed greater bone volume fraction, bone mineral density, tissue ingrowth, collagen density, and osteoblastic markers in bioreactor constructs cultured for 14 days compared to those cultured for 1 or 7 days, and acellular constructs. Taken together, these data demonstrate that culturing MSCs under perfusion culture for at least 14 days in vitro improves the quantity and quality of bone formation in vivo. This study highlights the need for optimizing in vitro bioreactor culture duration of engineered constructs to achieve the desired level of bone formation.
Pub.: 13 Sep '17, Pinned: 14 Oct '17
Abstract: We report engineering of half-centimeter-sized bone constructs created in vitro using human adipose-derived stem cells (hASCs), 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 diameter x 4 mm thick), 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 microm/s), via enhanced mass transport. After 5 weeks of cultivation, the addition of osteogenic supplements (dexamethasone, sodium-beta-glycerophosphate, and ascorbic acid-2-phosphate) to culture medium significantly increased the construct cellularity and the amounts of bone matrix components (collagen, bone sialoprotein, and 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.
Pub.: 15 Aug '09, Pinned: 17 Oct '17
Abstract: Identifying mechanisms by which cells of the osteoblastic lineage communicate in vivo is complicated by the mineralised matrix that encases osteocytes, and thus, vital mechanoadaptive processes used to achieve load-bearing integrity remain unresolved. We have used the coculture of immunomagnetically purified osteocytes and primary osteoblasts from both embryonic chick long bone and calvariae to examine these mechanisms. We exploited the fact that purified osteocytes are postmitotic to examine both their effect on proliferation of primary osteoblasts and the role of gap junctions in such communication. We found that chick long bone osteocytes significantly increased basal proliferation of primary osteoblasts derived from an identical source (tibiotarsi). Using a gap junction inhibitor, 18β-glycyrrhetinic acid, we also demonstrated that this osteocyte-related increase in osteoblast proliferation was not reliant on functional gap junctions. In contrast, osteocytes purified from calvarial bone failed to modify basal proliferation of primary osteoblast, but long bone osteocytes preserved their proproliferative action upon calvarial-derived primary osteoblasts. We also showed that coincubated purified osteocytes exerted a marked inhibitory action on mechanical strain-related increases in proliferation of primary osteoblasts and that this action was abrogated in the presence of a gap junction inhibitor. These data reveal regulatory differences between purified osteocytes derived from functionally distinct bones and provide evidence for 2 mechanisms by which purified osteocytes communicate with primary osteoblasts to coordinate their activity.
Pub.: 14 Jan '17, Pinned: 14 Oct '17
Abstract: Multiple factors contribute to bone loss in inflammatory diseases such as rheumatoid arthritis (RA), but circulating inflammatory factors and immobilization play a crucial role. Mechanical loading prevents bone loss in the general population, but the effects of mechanical loading in patients with RA are less clear. Therefore, we aimed to investigate whether mechanical stimuli reverse the stimulatory effect of RA serum on osteocyte-to-osteoclast communication. Human primary osteocytes were pretreated with 10 % RA serum or healthy control serum for 7 days, followed by 1 h ± mechanical loading by pulsating fluid flow (PFF). Nitric oxide (NO) and prostaglandin E2 were measured in the medium. Receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG), interleukin-6 (IL-6), cyclooxygenase-2 (COX2), matrix-extracellular phosphoglycoprotein (MEPE), cysteine-rich protein 61 (CYR61), and SOST gene expression was quantified by qPCR. Osteoclast precursors were cultured with PFF-conditioned medium (PFF-CM) or static-conditioned medium (stat-CM), and osteoclast formation was assessed. RA serum alone did not affect IL-6, CYR61, COX2, MEPE, or SOST gene expression in osteocytes. However, RA serum enhanced the RANKL/OPG expression ratio by 3.4-fold, while PFF nullified this effect. PFF enhanced NO production to the same extent in control serum (2.6-3.5-fold) and RA serum-pretreated (2.7-3.6-fold) osteocytes. Stat-CM from RA serum-pretreated osteocytes enhanced osteoclastogenesis compared with stat-CM from control serum-pretreated osteocytes, while PFF nullified this effect. In conclusion, RA serum, containing inflammatory factors, did not alter the intrinsic capacity of osteocytes to sense mechanical stimuli, but upregulated osteocyte-to-osteoclast communication. Mechanical loading nullified this upregulation, suggesting that mechanical stimuli could contribute to the prevention of osteoporosis in inflammatory disease.
Pub.: 15 May '15, Pinned: 14 Oct '17
Abstract: Bone remodelling is the continuous turnover of bone by resorption and formation. It is controlled by interstitial fluid flow sensed by osteocytes. The refilling of bone resorption sites has been shown to be curvature driven. In vitro, curvature influences tissue growth and cytoskeletal arrangements under static and perfused conditions. Nevertheless, this has only been demonstrated for non-mineralized tissue in limited three-dimensional volumes. This study aims at investigating the influence of three different channel curvatures (S, -2.00 mm(-1); M, -1.33 mm(-1); L, -0.67 mm(-1)) on mineralized tissue formation in three dimensions under static and perfused conditions. The ingrowth of mineralized tissue into the channels was dependent on curvature and was higher under perfusion (M and S channels). L channels were not closed in any group compared with partially (static) or fully (perfused) closed M and S channels. Mineralized tissue morphology was cortical-like in static samples and trabecular-like in perfused samples. Our results suggest that the three-dimensional in vitro model presented is not only able to reveal effects of curvature on mineralized tissue formation, but may be used as an in vitro model for critical size defects in trabecular or cortical bone.
Pub.: 14 Oct '16, Pinned: 14 Oct '17
Abstract: The aim of this study was to investigate the effects of perfusion co-culture on bone tissue regeneration in vitro. Human mesenchymal stem cell (hMSC)-derived osteoblasts and THP-1 human acute monocytic leukemia cell line-derived osteoclasts were dynamically co-cultured on the chitosan-hydroxyapatite (chitosan-HA) superporous hydrogel. In the perfusion bioreactor set-up, bidirectional recycled perfusion with 6 mL/h flow rate was applied and cell seeding was realized in two-steps with a preculture time of 12 days. Outcomes were compared to static cultures. Two-step cell seeding and long preculture ensured good adhesion of cells on the scaffold surface and minimized cell loss during perfusion. The perfusion bioreactor enhanced mass transfer throughout the scaffolds, thus increased cellularity and provided flow-induced mechanical stimulation for osteoblastic and osteoclastogenic differentiation. The results indicated that osteoblast and osteoclast co-cultures in perfusion bioreactors provide a one-step approach to in vitro bone tissue engineering and emphasized the significance of enhanced mass transfer and mechanical stimulation on cellular activity and differentiation.
Pub.: 10 May '16, Pinned: 14 Oct '17
Abstract: A review about design, manufacture, and mechanobiology of biodegradable scaffolds for bone tissue engineering is given. First, fundamental aspects about bone tissue engineering and considerations related to scaffold design are established. Second, issues related to scaffold biomaterials and manufacturing processes are discussed. Finally, mechanobiology of bone tissue and computational models developed for simulating how bone healing occurs inside a scaffold are described.
Pub.: 18 Apr '15, Pinned: 14 Oct '17
Abstract: Silk fibroin is an important polymer for scaffold designs, forming biocompatible and mechanically robust biomaterials for bone, cartilage, and ligament tissue engineering. In the present work, 3D biomaterial matrices were fabricated from silk fibroin with controlled pore diameter and pore interconnectivity, and utilized to engineer bone starting from human mesenchymal stem cells (hMSC). Osteogenic differentiation of hMSC seeded on these scaffolds resulted in extensive mineralization, alkaline phosphatase activity, and the formation of interconnected trabecular- or cortical-like mineralized networks as a function of the scaffold design utilized; allowing mineralized features of the tissue engineered bone to be dictated by the scaffold features used initially in the cell culture process. This approach to scaffold predictors of tissue structure expands the window of applications for silk fibroin-based biomaterials into the realm of directing the formation of complex tissue architecture. As a result of slow degradation inherent to silk fibroin, scaffolds preserved their initial morphology and provided a stable template during the mineralization phase of stem cells progressing through osteogenic differentiation and new extracellular matrix formation. The slow degradation feature also facilitated transport throughout the 3D scaffolds to foster improved homogeneity of new tissue, avoiding regions with decreased cellular density. The ability to direct bone morphology via scaffold design suggests new options in the use of biodegradable scaffolds to control in vitro engineered bone tissue outcomes.
Pub.: 24 May '07, Pinned: 17 Oct '17
Abstract: The aim of this study was to investigate the effect of three-dimensional silk fibroin scaffold preparation methods (aqueous and solvent) on osteogenic responses by human bone marrow stem cells (hMSCs). Macroporous 3D protein scaffolds with similar sized pores of 900+/-50 microm were prepared either by an organic solvent process (hexafluoro-2-propanol, HFIP) or an aqueous process. hMSCs were expanded, seeded on the scaffolds, and cultured up to 28 days under static conditions in osteogenic media. hMSCs seeded onto the water-based silk scaffolds showed a significant increase in cell numbers (p<0.01) vs. the HFIP-prepared silk scaffolds. Significantly higher (p<0.01) alkaline phosphatase (ALPase) activity and calcium deposition were apparent after 28 days of culture in the water-based silk scaffolds when compared to the HFIP-derived silk scaffolds. Transcript levels for collagen type I (Col I), ALP, and osteopontin (OP) increased (p<0.05) in the water-based silk scaffolds in comparison to the HFIP-derived materials. At early stages of culture, increased expression of OP and collagen type II (Col II) were also observed in both scaffolds. Expression of Col II, MMP 13, Col I, and OP proteins increased in the water-based silk scaffolds in comparison to the HFIP-derived scaffolds while bone sialoprotein (BSP) proteins increased in the HFIP-derived silk scaffolds in comparison to the water-based scaffolds after 28 days of culture. Histological analysis showed the development of bone-like trabeculae with cuboid cells in an extracellular matrix (ECM) in the water-based silk scaffolds with more organization than in the HFIP-derived material after 28 days of culture. Alcian blue staining demonstrated the presence of proteoglycan in the ECM formed in the water-based scaffolds but not in the HFIP-prepared silk scaffolds. The results suggest that macroporous 3D aqueous-derived silk fibroin scaffolds provide improved bone-related outcomes in comparison to the HFIP-derived systems. These data illustrate the importance of materials processing on biological outcomes, as the same protein, silk fibroin, was used in both preparations.
Pub.: 11 Feb '05, Pinned: 17 Oct '17
Abstract: Tissue engineering focuses on the repair and regeneration of tissues through the use of biodegradable scaffold systems that structurally support regions of injury while recruiting and/or stimulating cell populations to rebuild the target tissue. Within bone tissue engineering, the effects of scaffold architecture on cellular response have not been conclusively characterized in a controlled-density environment. We present a theoretical and practical assessment of the effects of polycaprolactone (PCL) scaffold architectural modifications on mechanical and flow characteristics as well as MC3T3-E1 preosteoblast cellular response in an in vitro static plate and custom-designed perfusion bioreactor model. Four scaffold architectures were contrasted, which varied in inter-layer lay-down angle and offset between layers, while maintaining a structural porosity of 60 ± 5%. We established that as layer angle was decreased (90° vs. 60°) and offset was introduced (0 vs. 0.5 between layers), structural stiffness, yield stress, strength, pore size, and permeability decreased, while computational fluid dynamics-modeled wall shear stress was increased. Most significant effects were noted with layer offset. Seeding efficiencies in static culture were also dramatically increased due to offset (∼ 45% to ∼ 86%), with static culture exhibiting a much higher seeding efficiency than perfusion culture. Scaffold architecture had minimal effect on cell response in static culture. However, architecture influenced osteogenic differentiation in perfusion culture, likely by modifying the microfluidic environment.
Pub.: 30 Jan '14, Pinned: 14 Oct '17
Abstract: Silk fibroin (SF) is a fibrous protein which is produced mainly by silkworms and spiders. Its unique mechanical properties, tunable biodegradation rate and the ability to support the differentiation of mesenchymal stem cells along the osteogenic lineage, have made SF a favorable scaffold material for bone tissue engineering. SF can be processed into various scaffold forms, combined synergistically with other biomaterials to form composites and chemically modified, which provides an impressive toolbox and allows SF scaffolds to be tailored to specific applications. This review discusses and summarizes recent advancements in processing SF, focusing on different fabrication and functionalization methods and their application to grow bone tissue in vitro and in vivo. Potential areas for future research, current challenges, uncertainties and gaps in knowledge are highlighted.Silk fibroin is a natural biomaterial with remarkable biomedical and mechanical properties which make it favorable for a broad range of bone tissue engineering applications. It can be processed into different scaffold forms, combined synergistically with other biomaterials to form composites and chemically modified which provides a unique toolbox and allows silk fibroin scaffolds to be tailored to specific applications. This review discusses and summarizes recent advancements in processing silk fibroin, focusing on different fabrication and functionalization methods and their application to grow bone tissue in vitro and in vivo. Potential areas for future research, current challenges, uncertainties and gaps in knowledge are highlighted.
Pub.: 12 Sep '15, Pinned: 14 Oct '17
Abstract: For bone tissue engineering synthetic biphasic calcium phosphate (BCP) with a hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ratio of 60/40 (BCP60/40) is successfully clinically applied, but the high percentage of HA may hamper efficient scaffold remodelling. Whether BCP with a lower HA/β-TCP ratio (BCP20/80) is more desirable is still unclear. Vascular development is needed before osteogenesis can occur. We aimed to test the osteogenic and/or vasculogenic differentiation potential as well as degradation of composites consisting of human adipose stem cells (ASCs) seeded on BCP60/40 or BCP20/80 incorporated in fibrin gels that trigger neovascularization for bone regeneration. ASC attachment to BCP60/40 and BCP20/80 within 30 min was similar (>93%). After 11 days of culture BCP20/80-based composites showed increased alkaline phosphatase activity and DMP1 gene expression, but not RUNX2 and osteonectin expression, compared to BCP60/40-based composites. BCP20/80-based composites also showed enhanced expression of the vasculogenic markers CD31 and VEGF189, but not VEGF165 and endothelin-1. Collagen-1 and collagen-3 expression was similar in both composites. Fibrin degradation was increased in BCP20/80-based composites at day 7. In conclusion, BCP20/80-based composites showed enhanced osteogenic and vasculogenic differentiation potential compared to BCP60/40-based composites in vitro, suggesting that BCP20/80-based composites might be more promising for in vivo bone augmentation than BCP60/40-based composites.
Pub.: 23 Aug '16, Pinned: 14 Oct '17
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