Biomimetic tubular nanofiber mesh and platelet rich plasma-mediated delivery of BMP-7 for large bone defect regeneration

There is a growing need for successful bone tissue engineering strategies and advanced biomaterials that mimic the structure and function of native tissues carry great promise. Successful bone repair approaches may include an osteoconductive scaffold, osteoinductive growth factors, cells with an osteogenic potential and capacity for graft vascularisation. To increase osteoinductivity of biomaterials, the local combination and delivery of growth factors has been developed. In the present study we investigated the osteogenic effects of calcium phosphate (CaP)-coated nanofiber mesh tube-mediated delivery of BMP-7 from a PRP matrix for the regeneration of critical sized segmental bone defects in a small animal model. Bilateral full-thickness diaphyseal segmental defects were created in twelve male Lewis rats and nanofiber mesh tubes were placed around the defect. Defects received either treatment with a CaP-coated nanofiber mesh tube (n?=?6), an un-coated nanofiber mesh tube (n=6) a CaP-coated nanofiber mesh tube with PRP (n=6) or a CaP-coated nanofiber mesh tube in combination with 5?μg?BMP-7 and PRP (n?=?6). After 12?weeks, bone volume and biomechanical properties were evaluated using radiography, microCT, biomechanical testing and histology. The results demonstrated significantly higher biomechanical properties and bone volume for the BMP group compared to the control groups. These results were supported by the histological evaluations, where BMP group showed the highest rate of bone regeneration within the defect. In conclusion, BMP-7 delivery via PRP enhanced functional bone defect regeneration, and together these data support the use of BMP-7 in the treatment of critical sized defects.     

Mechanical properties of a biodegradable bone regeneration scaffold

Poly (Propylene Fumarate) (PPF), a novel, bulk erosion, biodegradable polymer, has been shown to have osteoconductive effects in vivo when used as a bone regeneration scaffold (Peter, S. J., Suggs, L. J., Yaszemski, M. J., Engel, P. S., and Mikos, A. J., 1999, J. Biomater. Sci. Polym. Ed., 10, pp. 363-373). The material properties of the polymer allow it to be injected into irregularly shaped voids in vivo and provide mechanical stability as well as function as a bone regeneration scaffold. We fabricated a series of biomaterial composites, comprised of varying quantities of PPF, NaCl and beta-tricalcium phosphate (beta-TCP), into the shape of right circular cylinders and tested the mechanical properties in four-point bending and compression. The mean modulus of elasticity in compression (Ec) was 1204.2 MPa (SD 32.2) and the mean modulus of elasticity in bending (Eb) was 1274.7 MPa (SD 125.7). All of the moduli were on the order of magnitude of trabecular bone. Changing the level of NaCl from 20 to 40 percent, by mass, did not decrease Ec and Eb significantly, but did decrease bending and compressive strength significantly. Increasing the beta-TCP from 0.25 g/g PPF to 0.5 g/g PPF increased all of the measured mechanical properties of PPF/NVP composites. These results indicate that this biodegradable polymer composite is an attractive candidate for use as a replacement scaffold for trabecular bone.     

Osteogenic response to BMP-2 of hMSCs grown on apatite-coated scaffolds

Osteoconductive materials play a critical role in promoting integration with surrounding bone tissue and resultant bone repair in vivo. However, the impact of 3D osteoconductive substrates coupled with soluble signals on progenitor cell differentiation is not clear. In this study, we investigated the influence of bone morphogenetic protein-2 (BMP-2) concentration on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) when seeded in carbonated apatite-coated polymer scaffolds. Mineralized scaffolds were more hydrophilic and adsorbed more BMP-2 compared to non-mineralized scaffolds. Changes in alkaline phosphatase (ALP) activity within stimulated hMSCs were dependent on the dose of BMP-2 and the scaffold composition. We detected more cell-secreted calcium on mineralized scaffolds at all time points, and higher BMP-2 concentrations resulted in increased ALP and calcium levels. RUNX2 and IBSP gene expression within hMSCs was affected by both substrate and soluble signals, SP7 by soluble factors, and SPARC by substrate-mediated cues. The present data indicate that a combination of apatite and BMP-2 do not simply enhance the osteogenic response of hMSCs, but act through multiple pathways that may be both substrate- and growth factor-mediated. Thus, multiple signaling strategies will likely be necessary to achieve optimal bone regeneration.     

Design and manufacture of combinatorial calcium phosphate bone scaffolds

It is well known that pore design is an important determinant of both the quantity and distribution of regenerated bone in artificial bone tissue scaffolds. A requisite feature is that scaffolds must contain pore interconnections on the order of 100-1000 μm (termed macroporosity). Within this range, there is not a definitive optimal interconnection size. Recent results suggest that pore interconnections permeating the scaffold build material on the order of 2-20 μm (termed microporosity) drive bone growth into the macropore space at a faster rate and also provide a new space for bone growth, proliferating throughout the interconnected microporous network. The effects of microstructural features on bone growth has yet to be fully understood. This work presents the manufacture and characterization of novel combinatorial test scaffolds, scaffolds that test multiple microporosity and macroporosity designs within a single scaffold. Scaffolds such as this can efficiently evaluate multiple mechanical designs, with the advantage of having the designs colocated within a single defect site and therefore less susceptible to experimental variation. This paper provides the manufacturing platform, manufacturing control method, and demonstrates the manufacturing capabilities with three representative scaffolds.     

The growth and phenotypic expression of human osteoblasts

The care of patients with a skeletal deficiency currently involves the use of bone graft or a non-biologic material such as a metal or polymer. There are alternate possibilities in development which involve the growth of bone cells (osteoblasts) on degradable polymer scaffolds. These tissue engineering strategies require production of the polymeric scaffold, cellular harvest followed by either ex vivo or in vivo growth of the cells on the scaffold, and exploration of the interaction between the cell and scaffold. Research into these strategies utilizes cells from a variety of species, but clinical applications will likely require human osteoblasts. This study explores the process whereby human osteoblasts are harvested under sterile conditions during joint replacement surgery from normally discarded cancellous bone, transported from the operating room to the lab, and grown in culture. This process is feasible, and the cells express their phenotype via the production of alkaline phosphatase and collagen in culture.     

Preparation and Characterization of an Advanced Medical Device for Bone Regeneration

Tridimensional scaffolds can promote bone regeneration as a framework supporting the migration of cells from the surrounding tissue into the damaged tissue and as delivery systems for the controlled or prolonged release of cells, genes, and growth factors. The goal of the work was to obtain an advanced medical device for bone regeneration through coating a decellularized and deproteinized bone matrix of bovine origin with a biodegradable, biocompatible polymer, to improve the cell engraftment on the bone graft. The coating protocol was studied and set up to obtain a continuous and homogeneous polylactide-co-glycolide (PLGA) coating on the deproteinized bone matrix Orthoss® block without occluding pores and decreasing the scaffold porosity. The PLGA-coated scaffolds were characterized for their morphology and porosity. The effects of PLGA polymer coating on cell viability were assessed with the 3-(4,5-dimethyl-2-thiazolyl)-2,5 diphenyl-2H-tetrazolium assay. The polymer solution concentration and the number of polymeric layers were the main variables affecting coating efficiency and porosity of the original decellularized bone matrix. The designed polymer coating protocol did not affect the trabecular structure of the original decellularized bone matrix. The PLGA-coated decellularized bone matrix maintained the structural features, and it improved the ability in stimulating fibroblasts attachment and proliferation.     

Enhancement of in vivo bone regeneration efficacy of human mesenchymal stem cells

We investigated whether transplantation of osteogenically differentiated bone marrow-derived mesenchymal stem cells (BMMSCs) and the use of an hydroxyapatite (HAp) scaffold can enhance the in vivo bone formation efficacy of human BMMSCs. Three months after implantation to the subcutaneous dorsum of athymic mice, transplantation of osteogenically differentiated human BMMSCs increased the bone formation area and calcium deposition to 7.1- and 6.2-folds, respectively, of those of transplantation of undifferentiated BMMSCs. The use of the HAp scaffold increased the bone formation area and calcium deposition to 3.7- and 3.5-folds, respectively, of those of a polymer scaffold. Moreover, a combination of transplantation of osteogenically differentiated BMMSCs and HAp scaffold further increased the bone formation area and calcium deposition to 10.6- and 9.3-folds, respectively, of those of transplantation of undifferentiated BMMSCs seeded onto polymer scaffolds. The factorial experimental analysis showed that osteogenic differentiation of BMMSCs prior to transplantation has a stronger positive effect than the HAp scaffold on in vivo bone formation.     

Self-fitting shape memory polymer foam inducing bone regeneration: A rabbit femoral defect study

Although tissue engineering has been attracted greatly for healing of critical-sized bone defects, great efforts for improvement are still being made in scaffold design. In particular, bone regeneration would be enhanced if a scaffold precisely matches the contour of bone defects, especially if it could be implanted into the human body conveniently and safely. In this study, polyurethane/hydroxyapatite-based shape memory polymer (SMP) foam was fabricated as a scaffold substrate to facilitate bone regeneration. The minimally invasive delivery and the self-fitting behavior of the SMP foam were systematically evaluated to demonstrate its feasibility in the treatment of bone defects in vivo. Results showed that the SMP foam could be conveniently implanted into bone defects with a compact shape. Subsequently, it self-matched the boundary of bone defects upon shape-recovery activation in vivo. Micro-computed tomography determined that bone ingrowth initiated at the periphery of the SMP foam with a constant decrease towards the inside. Successful vascularization and bone remodeling were also demonstrated by histological analysis. Thus, our results indicate that the SMP foam demonstrated great potential for bone regeneration.     

Fabrication of individual scaffolds based on a patient-specific alveolar bone defect model

Fabricating individualized tissue engineering scaffolds based on the three-dimensional shape of patient bone defects is required for the successful clinical application of bone tissue engineering. However, there are currently no reported studies of individualized bone tissue engineering scaffolds that truly reproduce a patient-specific bone defect. We fabricated individualized tissue engineering scaffolds based on alveolar bone defects. The individualized poly(lactide-co-glycolide) and tricalcium phosphate composite scaffolds were custom-made by acquiring the three-dimensional model through computed tomography, which was input into the computer-aided low-temperature deposition manufacturing system. The three-dimensional shape of the fabricated scaffold was identical to the patient-specific alveolar bone defects, with an average macropore diameter of 380 μm, micropore diameters ranging from 3 to 5 μm, and an average porosity of 87.4%. The mechanical properties of the scaffold were similar to adult cancellous bone. Scaffold biocompatibility was confirmed by attachment and proliferation of human bone marrow mesenchymal stem cells. Successful realization of individualized scaffold fabrication will enable clinical application of tissue-engineered bone at an early date.     

Bone regeneration: in vitro evaluation of the behaviour of osteoblast-like MG63 cells placed in contact with polylactic-co-glycolic acid, deproteinized bovine bone and demineralized freeze-dried bone allograft

Insufficient bone density of the alveolar crests, caused by loss of the dental elements, sometimes impedes the primary stability of an integrated bone implant. The techniques of bone regeneration allow to obtain a sufficient quantity of alveolar bone to permit the implant rehabilitation of the edentulous crests. Today several grafting materials are available and they have different characteristics, according to their structure, which influence the different behaviour of the grafting materials to the bone and the implant surface. The aim of this study is to evaluate the interaction between a human osteosarcoma MG63 cell line and three different biomaterials: polylactic-co-glycolic acid (PLAGA), deproteinized bovine bone and demineralised freeze-dried bone allograft (DFDBA). From this study a different behaviour emerges of the osteoblast-like MG63 cells in relation to the sublayer on which these cells were placed in culture. The results of the study, in fact, demonstrate that the most osteoconductive material of the three analysed is the DFDBA, followed by DPBB. On the contrary, the PLGA, because of its roughness, does not seem to represent a valid support for cell growth, and does not encourage any morphologic modification in tumor cells. Furthermore, deproteinized bovine bone shows a differentiating effect which could lead to hypothesise an osteoconductive capacity of this biomaterial. Further studies should be carried out with the aim of explaining the results obtained.     

Biomineralization and size control of stable calcium phosphate core-protein shell nanoparticles: potential for vaccine applications

Calcium phosphate (CaP) polymorphs are nontoxic, biocompatible and hold promise in applications ranging from hard tissue regeneration to drug delivery and vaccine design. Yet, simple and robust routes for the synthesis of protein-coated CaP nanoparticles in the sub-100 nm size range remain elusive. Here, we used cell surface display to identify disulfide-constrained CaP binding peptides that, when inserted within the active site loop of Escherichia coli thioredoxin 1 (TrxA), readily and reproducibly drive the production of nanoparticles that are 50-70 nm in hydrodynamic diameter and consist of an approximately 25 nm amorphous calcium phosphate (ACP) core stabilized by the protein shell. Like bone and enamel proteins implicated in biological apatite formation, peptides supporting nanoparticle production were acidic. They also required presentation in a loop for high-affinity ACP binding as elimination of the disulfide bridge caused a nearly 3-fold increase in hydrodynamic diameters. When compared to a commercial aluminum phosphate adjuvant, the small core-shell assemblies led to a 3-fold increase in mice anti-TrxA titers 3 weeks postinjection, suggesting that they might be useful vehicles for adjuvanted antigen delivery to dendritic cells.     

Hydrogels and electrospun nanofibrous scaffolds of N-methylene phosphonic chitosan as bioinspired osteoconductive materials for bone grafting

In this work, N-methylene phosphonic chitosan (NMPC) based hydrogels and electrospun nanofibrous scaffolds are reported with objective to obtain osteoconductive and osteoinductive matrixes for bone grafting applications. NMPC, a phosphorylated derivative of chitosan, is known to mimic the function of non collagenous phosphoproteins in providing nucleation sites for biomineralization. NMPC hydrogels were prepared by crosslinking between NMPC and genipin. A detailed investigation of physicochemical properties of NMPC solutions is also carried out in order to obtain beads free nanofibers. Both NMPC gels and nanofibers were further evaluated for their biomineralization potential and biocompatibility with human osteoblast like cells. Results indicated that hydrogels and nanofibrous scaffolds NMPC are biocompatible and significantly osteoinductive compared to tissue culture plate controls. However, cells seeded on nanofibrous scaffolds exhibited greater proliferation measured by MTT assay, and higher expression of early markers for osteogenic differentiation proving the superior applicability of nanofibrous scaffolds for bone grafting applications.     

Evaluation of biological protein-based collagen scaffolds in cartilage and musculoskeletal tissue engineering--a systematic review of the literature

The term tissue engineering is the technology that combines cells, engineering and biological/synthetic material in order to repair, replace or regenerate biological tissues such as bone, muscle, tendons and cartilage. The major human applications of tissue engineering are: skin, bone, cartilage, corneas, blood vessels, left mainstem bronchus and urinary structures. In this systematic review several criteria were identified as the most desirable characteristics of an ideal scaffold. These state that an ideal scaffolds needs to be biodegradable, possess mechanical strength, be highly porous, biocompatible, non-cytotoxic, non antigentic, stuitable for cell attachment, proliferation and differentiation, flexible and elastic, three dimensional, osteoconductive and support the transport of nutrients and metabolic waste. Subsequently, studies reporting on the various advantages and disadvantages of using collagen based scaffolds in musculoskeletal and cartilage tissue engineering were identified. The purpose of this review is to 1) provide a list of ideal characteristics of a scaffold as identified in the literature 2) identify different types of biological protein-based collagen scaffolds used in musculoskeletal and cartilage tissue engineering 3) assess how many of the criteria each scaffold type meets 4) weigh different scaffolds against each other according to their relative properties and shortcomings. The rationale behind this approach is that the ideal scaffold material has not yet been identified. Hence, this review will define how many of the identified ideal characteristics are fulfilled by natural collagen-based scaffolds and address the shortcomings of its use as found in the literature.     

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.     

Synthesis of versatile thiol-reactive polymer scaffolds via RAFT polymerization

Well-defined polymer scaffolds convertible to (multi)functional polymer structures via selective and efficient modifications potentially provide an easy, versatile, and useful approach for a wide variety of applications. Considering this, a homopolymer scaffold, poly(pyridyldisulfide ethylmethacrylate) (poly(PDSM)), having pendant groups selectively reactive with thiols, was synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization. Soluble polymers with controlled molecular weights and narrow PDIs were generated efficiently. The versatility of the scaffold to generate random co- and ter-polymers combining multiple functionalities with controlled-composition was shown by separate and simultaneous conjugation of different mercapto-compounds, including a tripeptide in one-step. Conversion of water-insoluble scaffold to peptide-containing water-soluble copolymers was observed to yield nanometer-size particles with narrow polydispersity. The overall results suggest that the well-defined PDSM homopolymer scaffold generated via RAFT polymerization can be a versatile building block for generation of new structures having potential for drug delivery applications via a straightforward synthetic approach.     

Polyrotaxanes for applications in life science and biotechnology

Due to their low cytotoxicity, controllable size, and unique architecture, cyclodextrin (CD)-based polyrotaxanes and polypseudorotaxanes have inspired interesting exploitation as novel biomaterials. This review will update the recent progress in the studies on the structures of polyrotaxanes and polypseudorotaxanes based on different CDs and polymers, followed by summarizing their potential applications in life science and biotechnology, such as drug delivery, gene delivery, and tissue engineering. CD-based biodegradable polypseudorotaxane hydrogels could be used as promising injectable drug delivery systems for sustained and controlled drug release. Polyrotaxanes with drug or ligand-conjugated CDs threaded on polymer chain with biodegradable end group could be useful for controlled and multivalent targeting delivery. Cationic polyrotaxanes consisting of multiple oligoethylenimine-grafted CDs threaded on a block copolymer chain were attractive non-viral gene carries due to the strong DNA-binding ability, low cytotoxicity, and high gene transfection efficiency. Cytocleavable end caps were also introduced in the polyrotaxane systems in order to ensure efficient endosomal escape for intracellular trafficking of DNA. Finally, hydrolyzable polyrotaxane hydrogels with cross-linked α-CDs could be a desirable scaffold for cartilage and bone tissue engineering.     

Interactions between human osteoblasts and prostate cancer cells in a novel 3D in vitro model

Cell-cell and cell-matrix interactions play a major role in tumor morphogenesis and cancer metastasis. Therefore, it is crucial to create a model with a biomimetic microenvironment that allows such interactions to fully represent the pathophysiology of a disease for an in vitro study. This is achievable by using three-dimensional (3D) models instead of conventional two-dimensional (2D) cultures with the aid of tissue engineering technology. We are now able to better address the complex intercellular interactions underlying prostate cancer (CaP) bone metastasis through such models. In this study, we assessed the interaction of CaP cells and human osteoblasts (hOBs) within a tissue engineered bone (TEB) construct. Consistent with other in vivo studies, our findings show that intercellular and CaP cell-bone matrix interactions lead to elevated levels of matrix metalloproteinases, steroidogenic enzymes and the CaP biomarker, prostate specific antigen (PSA); all associated with CaP metastasis. Hence, it highlights the physiological relevance of this model. We believe that this model will provide new insights for understanding of the previously poorly understood molecular mechanisms of bone metastasis, which will foster further translational studies, and ultimately offer a potential tool for drug screening.     

Interactions between human osteoblasts and prostate cancer cells in a novel 3D in vitro model

Cell-cell and cell-matrix interactions play a major role in tumor morphogenesis and cancer metastasis. Therefore, it is crucial to create a model with a biomimetic microenvironment that allows such interactions to fully represent the pathophysiology of a disease for an in vitro study. This is achievable by using three-dimensional (3D) models instead of conventional two-dimensional (2D) cultures with the aid of tissue engineering technology. We are now able to better address the complex intercellular interactions underlying prostate cancer (CaP) bone metastasis through such models. In this study, we assessed the interaction of CaP cells and human osteoblasts (hOBs) within a tissue engineered bone (TEB) construct. Consistent with other in vivo studies, our findings show that intercellular and CaP cell-bone matrix interactions lead to elevated levels of matrix metalloproteinases, steroidogenic enzymes and the CaP biomarker, prostate specific antigen (PSA); all associated with CaP metastasis. Hence, it highlights the physiological relevance of this model. We believe that this model will provide new insights for understanding of the previously poorly understood molecular mechanisms of bone metastasis, which will foster further translational studies, and ultimately offer a potential tool for drug screening.     

Integrin-supported fast rate intracellular delivery of plasmid DNA by extracellular matrix protein embedded calcium phosphate complexes

Extracellular matrix (ECM) proteins, such as collagen and fibronectin, play vital roles in development and maintenance of hard tissue (bone or tooth) and are, consequently, thoroughly investigated for construction of biomimetic scaffolds in combination with calcium phosphate (CaP) material (the major component of hard tissue) for bone or dental tissue engineering. Realizing the natural affinity of ECM components toward inorganic constituents of hard tissue, we successfully constructed the nanohybrids of DNA/CaP particles with either collagen 1 or fibronectin, which finally possessed the capability of specific recognition of integrin receptor for being swiftly internalized across the plasma membrane, leading to remarkably high transgene expression in mammalian cells. This new approach with precise receptor-specific delivery as well as 10- to 50-fold enhanced efficiency level compared to the classical one, has immediate applications for basic research and large scale production of recombinant therapeutic proteins and looks promising for gene therapy.     

Role of Wnts in prostate cancer bone metastases

Prostate cancer (CaP) is unique among all cancers in that when it metastasizes to bone, it typically forms osteoblastic lesions (characterized by increased bone production). CaP cells produce many factors, including Wnts that are implicated in tumor-induced osteoblastic activity. In this prospectus, we describe our research on Wnt and the CaP bone phenotype. Wnts are cysteine-rich glycoproteins that mediate bone development in the embryo and promote bone production in the adult. Wnts have been shown to have autocrine tumor effects, such as enhancing proliferation and protecting against apoptosis. In addition, we have recently identified that CaP-produced Wnts act in a paracrine fashion to induce osteoblastic activity in CaP bone metastases. In addition to Wnts, CaP cells express the soluble Wnt inhibitor dickkopf-1 (DKK-1). It appears that DKK-1 production occurs early in the development of skeletal metastases, which results in masking of osteogenic Wnts, thus favoring osteolysis at the metastatic site. As metastases progress, DKK-1 expression decreases allowing for unmasking of Wnt's osteoblastic activity and ultimately resulting in osteosclerosis at the metastatic site. We believe that DKK-1 is one of the switches that transitions the CaP bone metastasis activity from osteolytic to osteoblastic. Wnt/DKK-1 activity fits a model of CaP-induced bone remodeling occurring in a continuum composed of an osteolytic phase, mediated by receptor activator of NFkB ligand (RANKL), parathyroid hormone-related protein (PTHRP) and DKK-1; a transitional phase, where environmental alterations promote expression of osteoblastic factors (Wnts) and decreases osteolytic factors (i.e., DKK-1); and an osteoblastic phase, in which tumor growth-associated hypoxia results in production of vascular endothelial growth factor and endothelin-1, which have osteoblastic activity. This model suggests that targeting both osteolytic activity and osteoblastic activity will provide efficacy for therapy of CaP bone metastases.