Functional Bone Tissue Engineering Research Articles

Rapid biomimetic mineralization of keratin scaffold via electrodeposition with potential for bone regeneration

For bone regeneration, rapid mineralization is of particular importance in the preparation of biomimetic bone scaffolds, as for avoiding the drawbacks of prolonged mineralization which will cause the loss of growth factors and degradation. In this research, we developed a relatively rapid mineralization approach for constructing biomimetic keratin scaffold that exhibited highly interconnected pore and natural bone mimetic calcium phosphate coating by applying an electrodeposition technique. Mineralized keratin scaffold was obtained by freeze drying followed by electrodeposition for rapid biomimetic mineralization. The mineral coating morphology, component and crystal structure could be controllably tailored by manipulating the deposition electrode, voltage and duration. A satisfying coating of apatite layer on keratin scaffold could be obtained within a couple of hours by electrodeposition. By increasing the voltage and duration, a more favored amount of apatite coating which dominated by HA crystal could be formed. In addition, cell regeneration assay showed that mineralized biomimetic keratin scaffold exhibited more suitable supporting platform for the proliferation and osteoblastic differentiation of MC3T3 cell over pure keratin scaffold. Thus, the rapid electrodeposition mineralization approach presented in this work could be highly desired for fabricating biomimetic scaffolds in which biological molecules were loaded for functional bone tissue engineering applications.

A Hierarchical 3D Graft Printed with Nanoink for Functional Craniofacial Bone Restoration

AbstractAn ideal craniofacial bone repair graft shall not only focus on the repair ability but also the regeneration of natural architecture with occlusal loads‐related function restoration. However, such functional bone tissue engineering scaffold has rarely been reported. Herein, a hierarchical 3D graft is proposed for rebuilding craniofacial bone with both natural structure and healthy biofunction reconstruction. Inspired by the bone healing process, an organic–inorganic nanoink with ultrasmall calcium phosphate oligomers and bone morphogenetic protein‐2 incorporated is developed for spatiotemporal guidance of new bone. Based on such homogeneous nanoink, a biomimetic graft, including a cortical layer containing Haversian system, and a cancellous layer featured with triply periodic minimum surface macrostructures, is fabricated via projection‐based 3D printing method, and the layers are loaded with distinct concentrations of bioactive factors for regenerating new bone with gradient density. The graft exhibits excellent osteogenic and angiogenic potential in vitro, and accelerates revascularization and reconstructs neo‐bone with original morphology in vivo. Benefiting from such natural architecture, loading force is widely transferred with reduced stress concentration around the inserted dental implant. Taken from native physiochemical and structural cues, this wstudy provides a novel strategy for functional tissue engineering through designing function‐oriented biomaterials.

MicroRNA-loaded biomaterials for osteogenesis.

The large incidence of bone defects in clinical practice increases not only the demand for advanced bone transplantation techniques but also the development of bone substitute materials. A variety of emerging bone tissue engineering materials with osteogenic induction ability are promising strategies for the design of bone substitutes. MicroRNAs (miRNAs) are a class of non-coding RNAs that regulate intracellular protein expression by targeting the non-coding region of mRNA3′-UTR to play an important role in osteogenic differentiation. Several miRNA preparations have been used to promote the osteogenic differentiation of stem cells. Therefore, multiple functional bone tissue engineering materials using miRNA as an osteogenic factor have been developed and confirmed to have critical efficacy in promoting bone repair. In this review, osteogenic intracellular signaling pathways mediated by miRNAs are introduced in detail to provide a clear understanding for future clinical treatment. We summarized the biomaterials loaded with exogenous cells engineered by miRNAs and biomaterials directly carrying miRNAs acting on endogenous stem cells and discussed their advantages and disadvantages, providing a feasible method for promoting bone regeneration. Finally, we summarized the current research deficiencies and future research directions of the miRNA-functionalized scaffold. This review provides a summary of a variety of advanced miRNA delivery system design strategies that enhance bone regeneration.

Physico-chemical and biological properties of electrospun poly-L-lactic acid/hydroxyapatite nano-composite scaffold for bone regeneration

The main goal of tissue engineering is to design scaffolds with the suitable microenvironment, prominent mechanical properties, adequate biocompatibility, and biodegradability. In the present study, Poly-L-lactic acid (PLLA)/10% hydroxyapatite (HA) nano-composite (PLLA/HA) scaffold and pure PLLA scaffold were fabricated via electrospinning technique. They were then assessed for their potential as substrates for the cell culture. The scaffolds were characterized by scanning electron microscope, thermogravimetric analysis, differential thermal analysis, and fourier transform infrared spectroscopy. The results showed the dispersion and integration of HA nanoparticles in the composite scaffold. Pore size distribution measurements indicated that the scaffold pore size in terms of Ferret diameter varies between 0.1 and 22.0 µm. In vitro degradation behavior of the scaffold in phosphate buffered saline (PBS) was obtained by measuring water uptake, weight loss, and pH change of the PBS during the degradation period. We observed that the degradation of the scaffold could be markedly accelerated by HA nanoparticle (10%). It was also shown that acidification of the PBS due to the PLLA degradation is suppressed by the HA nanoparticles. Finally, human umbilical-cord-derived mesenchymal stem cells (hUC-MSCs) were used to evaluate the cellular support of the composite scaffolds. The presence of HA nanoparticles in the PLLA fibrous scaffolds resulted in promoting the attachment and the proliferation of the hUC-MSCs. This study suggests that the PLLA/HA scaffold could be the material of choice for functional bone tissue engineering.

Progress in orthopedic biomaterials and drug delivery

Musculoskeletal conditions, including arthritis, back pain, and skeletal injuries/trauma, affect 50 % of the adult population in the U.S.A., and account for the second greatest cause of disability worldwide (www.bjdonline.org). The aggregate total annual cost of musculoskeletal diseases was $796 billion in 2011. While most musculoskeletal injuries and diseases have lowmortality, they have a tremendous impact of quality of life and morbidity. Biomaterials have a long history in orthopedics—metal and plastic implants have been used over the last 60 years to treat end-stage joint degeneration and skeletal tissue loss, and alleviate the resulting pain and disability. First-generation orthopedic implants were designed to be biologically inert and selected primarily for their mechanical properties. However, they do not replicate the function of normal tissue and have a limited life span in the body, which is especially problematic in younger patients. Recent decades have focused on the development of biomaterials that are biodegradable and incorporate tissue-specific physicochemical cues to guide cell processes and tissue morphogenesis, with the goal of restoring natural tissue structure and function. These advances in biomaterials provide critical enabling technologies for the delivery of stem cells, gene therapies, and new biologics in the orthopedic field. This special issue highlights the recent advances and future directions in orthopedic biomaterials and drug delivery systems, and showcases emerging pharmaceutical and regenerative approaches to treat injuries, diseases, and disorders of the musculoskeletal system. Non-union bone fractures are a significant clinical challenge, and bone tissue engineering could have a key impact in improving current clinical options. Telvin et al. review the application of stem cells from various sources towards bone tissue engineering. The ability of transplanted stem cells to survive, continue to differentiate, and form bone tissue is key to making stem cell-mediated bone tissue regeneration approaches successful. The research article by Wen et al. precisely address this challenge. Their study compares the in vivo bone tissue forming ability of human bone marrow derived stem cells and human embryonic stem cells using osteoinductive mineralized materials as a scaffold. The article by Nyberg et al. reviews progress and challenges in achieving precise temporal and spatial control of growth factor presentation for functional bone tissue engineering ex vivo and in vivo. Ultimately, successful bone regeneration requires integration between the repair and native bone tissue. Botchway and colleagues address this critical issue, by modifying the surface of a bone allograft with FTY720, a synthetic analog of sphingosine 1-phosphate, to promote endogenous bone formation and osseous integration at the interface. Another key hurdle for the success of bone tissue engineering is vascularization in vivo. The review by Garcia and Garcia summarizes the current strategies and new developments targeted at vascularization of engineered bone, including growth factor and cell delivery, gene therapy, biomaterials, and combination approaches. The article by Tonello et al. demonstrates the potential of human placental matrix for promoting angiogenesis and its controlled release from biodegradable microparticles. In addition to bone, this special issue contains articles that highlight advances in cartilage tissue regeneration and osteoarthritis (OA) therapies. Osteoarthritis is the leading cause of * Blanka Sharma blanka.sharma@bme.ufl.edu

Boron containing poly-(lactide-co-glycolide) (PLGA) scaffolds for bone tissue engineering

Scaffold-based bone defect reconstructions still face many challenges due to their inadequate osteoinductive and osteoconductive properties. Various biocompatible and biodegradable scaffolds, combined with proper cell type and biochemical signal molecules, have attracted significant interest in hard tissue engineering approaches. In the present study, we have evaluated the effects of boron incorporation into poly-(lactide-co-glycolide-acid) (PLGA) scaffolds, with or without rat adipose-derived stem cells (rADSCs), on bone healing in vitro and in vivo. The results revealed that boron containing scaffolds increased in vitro proliferation, attachment and calcium mineralization of rADSCs. In addition, boron containing scaffold application resulted in increased bone regeneration by enhancing osteocalcin, VEGF and collagen type I protein levels in a femur defect model. Bone mineralization density (BMD) and computed tomography (CT) analysis proved that boron incorporated scaffold administration increased the healing rate of bone defects. Transplanting stem cells into boron containing scaffolds was found to further improve bone-related outcomes compared to control groups. Additional studies are highly warranted for the investigation of the mechanical properties of these scaffolds in order to address their potential use in clinics. The study proposes that boron serves as a promising innovative approach in manufacturing scaffold systems for functional bone tissue engineering.

Cyclic Tensile Strain Enhances Osteogenesis and Angiogenesis in Mesenchymal Stem Cells from Osteoporotic Donors

We have shown that the uniaxial cyclic tensile strain of magnitude 10% promotes and enhances osteogenesis of human mesenchymal stem cells (hMSC) and human adipose-derived stem cells (hASC) from normal, nonosteoporotic donors. In the present study, MSC from osteoporotic donors were analyzed for changes in mRNA expression in response to 10% uniaxial tensile strain to identify potential mechanisms underlying the use of this mechanical loading paradigm for prevention and treatment of osteoporosis. Human MSC isolated from three female, postmenopausal osteoporotic donors were analyzed for their responses to mechanical loading using microarray analysis of over 47,000 gene probes. Human MSC were seeded in three-dimensional collagen type I constructs to mimic the organic extracellular matrix of bone and 10% uniaxial cyclic tensile strain was applied to promote osteogenesis. Seventy-nine genes were shown to be regulated within hMSC from osteoporotic donors in response to 10% cyclic tensile strain. Upregulation of six genes were further confirmed with real-time RT-PCR: jun D proto-oncogene (JUND) and plasminogen activator, urokinase receptor (PLAUR), two genes identified as potential key molecules from network analysis; phosphoinositide-3-kinase, catalytic, delta polypeptide (PIK3CD) and wingless-type MMTV integration site family, member 5B (WNT5B), two genes with known importance in bone biology; and, PDZ and LIM domain 4 (PDLIM4) and vascular endothelial growth factor A (VEGFA), two genes that we have previously shown are significantly regulated in hASC in response to this mechanical stimulus. Function analysis indicated that 10% cyclic tensile strain induced expression of genes associated with cell movement, cell proliferation, and tissue development, including development in musculoskeletal and cardiovascular systems. Our results demonstrate that hMSC from aged, osteoporotic donors are capable of enhanced osteogenic differentiation in response to 10% cyclic tensile strain with significant increases in the expression of genes associated with enhanced cell proliferation, musculoskeletal development, and angiogenesis. Surprisingly, cyclic tensile strain of magnitude 10% not only enhanced osteogenesis in hMSC from osteoporotic donors, but also enhanced expression of angiogenic factors. Better understanding and methodologies to promote osteogenesis in hMSC from elderly, osteoporotic donors may greatly facilitate achieving long-term success in bone regeneration and functional bone tissue engineering for this ever-growing patient population.

Evaluation of three‐dimensional porous chitosan–alginate scaffolds in rat calvarial defects for bone regeneration applications

This study investigated the use of three-dimensional porous chitosan-alginate (CA) scaffolds for critical size calvarial defect (diameter, 5.0 mm) repair in Sprague-Dawley rats. CA scaffolds have been used for in vitro culture of many cell types and demonstrated osteogenesis in ectopic locations in vivo, but have yet to be evaluated for functional bone tissue engineering applications. CA scaffolds demonstrated the ability to support undifferentiated mesenchymal stem cells (MSCs) in culture for 14 days in vitro and promoted spherical morphology. In vivo tests were performed using CA scaffolds and CA scaffolds with treatments including undifferentiated MSCs, bone marrow aspirate, and bone morphogenetic protein-2 (BMP-2) growth factor in comparison to unfilled bone defect used as a control. The samples were analyzed with MicroCT, histology, and immunohistochemical staining at 4 and 16 weeks. Partial defect closure was observed in all experimental groups at 16 weeks, with the greatest defect closure (71.56 ± 19.74%) in the animal group treated with CA scaffolds with BMP-2 (CA + BMP-2). The experimental samples demonstrated osteogenesis in histology and immunohistochemical staining, with the CA + BMP-2 group, showing the greatest level of osteogenesis. Tissue engineered CA scaffolds show promise in reconstruction of critical size bone defects.

Comparative study of the osteogenic differentiation capacity of human bone marrow- and human adipose-derived stem cells under cyclic tensile stretch using quantitative analysis

Studies comparing the osteogenic differentiation capacity of human bone marrow-derived stem cells (hBMSCs) and human adipose-derived stem cells (hASCs) cultured in osteogenic differentiation medium have been inconclusive. Apart from chemical stimuli, mechanical stimuli have also been shown to be important in bone tissue engineering, which is referred to as functional bone tissue engineering. hBMSCs and hASCs have been shown to be sensitive to both chemical and mechanical stimuli. In an attempt to find a better seed cell in functional bone tissue engineering, we tried to quantify the osteogenic differentiation capacity of hBMSCs and hASCs under both mechanical and chemical stimuli. In this study, hBMSCs and hASCs were isolated from the same volunteers. Cells were cultured in osteogenic differentiation medium with and without exposure to cyclic tensile stretch (CTS). Quantitative measurement of alkaline phosphatase (ALP) activity revealed that the osteogenic differentiation capacity of hBMSCs was similar to that of hASCs in the early phase of differentiation in the CTS-stimulated groups. Quantitative measurement of mineralization showed that the late-phase osteogenic differentiation capacity of the hBMSCs was superior to that of hASCs in the CTS-stimulated groups. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed 5 and 10 days after cell culture. The results of the RT-PCR revealed that the osteogenic differentiation capacity of hASCs was inferior to that of hBMSCs both in the CTS-stimulated and unstimulated groups. All the results showed that both hBMSCs and hASCs were sensitive to CTS during the osteogenic differentiation process. This study compared the osteogenic differentiation capacity of hBMSCs and hASCs in response to mechanical stimulations and has important implications for the use of stem cells in functional bone tissue engineering and regenerative medicine.

Bone Tissue Engineering: Recent Advances and Challenges

The worldwide incidence of bone disorders and conditions has trended steeply upward and is expected to double by 2020, especially in populations where aging is coupled with increased obesity and poor physical activity. Engineered bone tissue has been viewed as a potential alternative to the conventional use of bone grafts, due to their limitless supply and no disease transmission. However, bone tissue engineering practices have not proceeded to clinical practice due to several limitations or challenges. Bone tissue engineering aims to induce new functional bone regeneration via the synergistic combination of biomaterials, cells, and factor therapy. In this review, we discuss the fundamentals of bone tissue engineering, highlighting the current state of this field. Further, we review the recent advances of biomaterial and cell-based research, as well as approaches used to enhance bone regeneration. Specifically, we discuss widely investigated biomaterial scaffolds, micro- and nano-structural properties of these scaffolds, and the incorporation of biomimetic properties and/or growth factors. In addition, we examine various cellular approaches, including the use of mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs), and platelet-rich plasma (PRP), and their clinical application strengths and limitations. We conclude by overviewing the challenges that face the bone tissue engineering field, such as the lack of sufficient vascularization at the defect site, and the research aimed at functional bone tissue engineering. These challenges will drive future research in the field.

Rapid mineralization of porous gelatin scaffolds by electrodeposition for bone tissue engineering

In bone tissue engineering, rapid mineralization of polymeric scaffolds is of particular importance in protecting the encapsulated therapeutic drugs or growth factors from loss and degradation. Here, we present a simple and rapid approach to the fabrication of mineralized porous scaffolds for bone tissue engineering. In this approach, three-dimensional (3-D) porous gelatin scaffolds were firstly fabricated by freeze-drying followed by an electrodeposition process for mineralization. We show that a high-quality apatite coating on the gelatin scaffold could be achieved within a couple of hours by electrodeposition. Increasing the deposition voltage or electrolyte temperature favored to the formation of large amounts of apatite coatings with compositions dominated by the hydroxyapatite crystals, whereas the presence of ultrasonic field facilitated the production of homogeneous apatite coatings. Moreover, biological assays indicated that the mineralized scaffolds exhibited better support for the proliferation and osteoblastic differentiation of MC3T3-E1 cells over a neat gelatin scaffold, especially for the case of mineralized scaffolds by electrodeposition at 60 °C. Therefore, the method developed would be highly desired for the rapid mineralization of polymer scaffolds in which biological molecules were loaded for functional bone tissue engineering applications.

Functionalization of poly(caprolactone) scaffolds by the surface mineralization for use in bone regeneration

The surface of a synthetic biopolymer scaffold was tailored by a mineralization with calcium phosphate for use as a functional bone tissue engineering matrix. Poly(ε-caprolactone) scaffold with a defined pore configuration constructed by a robocasting method was treated in a series of solutions involving steps of surface activation and calcium phosphate induction. The scaffold surface was completely covered with calcium phosphate nanocrystallites that had typical characteristics of bone mineral-like carbonate apatite. The scaffold with mineralized-surface demonstrated to support more favorable bone cell responses, including initial cell adhesion and proliferation and to allow higher loading of protein than the untreated-scaffold. The results suggest the developed scaffold has the potential for use as a bone regenerative matrix.

Microarray Analysis of Human Adipose-Derived Stem Cells in Three-Dimensional Collagen Culture: Osteogenesis Inhibits Bone Morphogenic Protein and Wnt Signaling Pathways, and Cyclic Tensile Strain Causes Upregulation of Proinflammatory Cytokine Regulators and Angiogenic Factors

Human adipose-derived stem cells (hASC) have shown great potential for bone tissue engineering. However, the molecular mechanisms underlying this potential are not yet known, in particular the separate and combined effects of three-dimensional (3D) culture and mechanical loading on hASC osteogenesis. Mechanical stimuli play a pivotal role in bone formation, remodeling, and fracture repair. To further understand hASC osteogenic differentiation and response to mechanical stimuli, gene expression profiles of proliferating or osteogenically induced hASC in 3D collagen I culture in the presence and absence of 10% uniaxial cyclic tensile strain were examined using microarray analysis. About 847 genes and 95 canonical pathways were affected during osteogenesis of hASC in 3D culture. Pathway analysis indicated the potential roles of Wnt/β-catenin signaling, bone morphogenic protein (BMP) signaling, platelet-derived growth factor (PDGF) signaling, and insulin-like growth factor 1 (IGF-1) signaling in hASC during osteogenic differentiation. Application of 10% uniaxial cyclic tensile strain suggested synergistic effects of strain with osteogenic differentiation media on hASC osteogenesis as indicated by significantly increased calcium accretion of hASC. There was no significant further alteration in the four major pathways (Wnt/β-catenin, BMP, PDGF, and IGF-1). However, 184 transcripts were affected by 10% cyclic tensile strain. Function and network analysis of these transcripts suggested that 10% cyclic tensile strain may play a role during hASC osteogenic differentiation by upregulating two crucial factors in bone regeneration: (1) proinflammatory cytokine regulators interleukin 1 receptor antagonist and suppressor of cytokine signaling 3; (2) known angiogenic inductors fibroblast growth factor 2, matrix metalloproteinase 2, and vascular endothelial growth factor A. This is the first study to investigate the effects of both 3D culture and mechanical load on hASC osteogenic differentiation. A complete microarray analysis investigating both the separate effect of soluble osteogenic inductive factors and the combined effects of chemical and mechanical stimulation was performed on hASC undergoing osteogenic differentiation. We have identified specific genes and pathways associated with mechanical response and osteogenic potential of hASC, thus providing significant information toward improved understanding of our use of hASC for functional bone tissue engineering applications.

Adipose-Derived Stem Cells in Functional Bone Tissue Engineering: Lessons from Bone Mechanobiology

This review aims to highlight the current and significant work in the use of adipose-derived stem cells (ASC) in functional bone tissue engineering framed through the bone mechanobiology perspective. Over a century of work on the principles of bone mechanosensitivity is now being applied to our understanding of bone development. We are just beginning to harness that potential using stem cells in bone tissue engineering. ASC are the primary focus of this review due to their abundance and relative ease of accessibility for autologous procedures. This article outlines the current knowledge base in bone mechanobiology to investigate how the knowledge from this area has been applied to the various stem cell-based approaches to engineering bone tissue constructs. Specific emphasis is placed on the use of human ASC for this application.

Mesenchymal stem cell‐seeded collagen matrices for bone repair: Effects of cyclic tensile strain, cell density, and media conditions on matrix contraction in vitro

Type I collagen is the most abundant extracellular matrix protein in bone and contains arginine- glycine-aspartic acid sequences that promote cell adhesion and proliferation. We have previously shown that human mesenchymal stem cells (hMSCs) seeded in three-dimensional (3D) collagen gels upregulate BMP-2 mRNA expression in response to tensile strain, indicative of osteogenesis. Therefore, collagen could be a promising scaffold material for functional bone tissue engineering using hMSCs. However, high contraction of the collagen gels by hMSCs poses a challenge to creating large, tissue-engineered bone constructs. The effects of cyclic tensile strain, medium (with and without dexamethasone), and hMSC seeding density on contraction of collagen matrices have not been investigated. hMSCs were seeded in 3D collagen gels and subjected to cyclic tensile strain of 10% or 12% for 4 h/day at a frequency of 1 Hz in osteogenic-differentiating or complete MSC growth media for up to 14 days. Viability of hMSCs was not affected by strain or media conditions. While initial seeding density affected matrix contraction alone, there was a high interdependence of strain and medium on matrix contraction. These findings suggest a correlation between hMSC proliferation and osteogenic differentiation on collagen matrix contraction that is affected by media, cell-seeding density, and cyclic tensile strain. It is vital to understand the effects of culture conditions on collagen matrix contraction by hMSCs in order to consider hMSC-seeded collagen constructs for functional bone tissue engineering in vitro.

Functional bone tissue engineering

Functional bone tissue engineering