Enhanced endothelial differentiation of adipose-derived stem cells by substrate nanotopography.

Adipose-derived stem cells (ADSCs) have great potential as a cell source for tissue engineering and regenerative medicine because they are easier to obtain, have lower donor-site morbidity and are available in larger numbers than stem cells harvested using bone marrow aspiration. Until now, little has been known about how nanotopography affects the proliferation and endothelial differentiation of ADSCs. In the present study, two nanograting substrates with a period (ridge and groove) of about 250 and 500 nm, respectively, were fabricated on quartz and their effect on ADSC fate was investigated. The results showed that proliferation of ADSCs on nanograting substrates decreased while cell attachment was not significantly affected compared to a flat substrate. Endothelial differentiation of ADSCs on both flat and nanograting substrates can be induced with vascular endothelial growth factor, as shown by immunofluorescent staining. Quantitative real-time PCR analysis showed significantly enhanced upregulation of vWF, PECAM-1 and VE-cadherin at the gene level by ADSCs on the nanograting substrates. In vitro angiogenesis assay on Matrigel showed that nanograting substrates enhanced capillary tube formation. This study highlights the beneficial influence of nanotopography on the differentiation of ADSC into endothelial cells which play an important role in vascularization.

Accelerated bone growth in vitro by the conjugation of BMP2 peptide with hydroxyapatite on titanium alloy.

Titanium alloys have been widely used in orthopedic practice due to their inherent bioactivity, however it is still insufficient to truly and reliably incorporate into living bone. In this work, polydopamine film was employed to induce the growth of hydroxyapatite (HA) on titanium alloy to enhance its osteoconductivity. Bone morphogenetic protein-2 (BMP2) peptide was absorbed into the HA particles for osteoinductivity. The precipitation of HA and the existence of BMP2 peptide were examined by X-ray diffraction, X-ray photoelectron spectroscopy and fluorescence microscopy. The dissolution of HA and the release of BMP2 peptide were monitored by measuring the concentrations of calcium ions and BMP2 peptide in phosphate buffered saline solution, respectively. The effect of BMP2 peptide incorporated into HA coating on bone growth was evaluated in vitro by cell culture tests, including cell attachment, alkaline phosphatase (ALP) activity, and gene expression. The results show that the HA particles grown on the substrate are mediated by the polydopamine film. The BMP2 peptide is distributed uniformly on HA-coated substrate and released in a sustained manner. Moreover, the conjunction of HA and BMP2 peptide increases cell adhesion, ALP activity and gene expression of osteogenic markers, which are potentially useful in the development of enhanced orthopedic medical devices.

Anti-fibrosis effect of BMP-7 peptide functionalization on cobalt chromium alloy.

Orthopedic metallic prosthetic implants are commonly made of cobalt chromium (CoCr) alloys. However, such metal-based implants are susceptible to fibrous capsule formation on the implant surface after implantation. At the bone-implant interface, this capsule can prevent implant integration, resulting in loosening and failure. Minimizing the development of such a capsule on the CoCr surface would improve direct bone-implant bonding leading to long-term implant functionality. We evaluated the anti-fibrosis effect of bone morphogenic protein-7 (BMP-7) peptide covalently bonded to CoCr alloy. This peptide, a biomimetic derivation of the knuckle epitope of BMP-7, was conjugated at the N-terminus with a cysteine amino acid. X-ray photoelectron spectroscopy (XPS) and probe binding assay were used to evaluate different stages of grafting and surface functionalization using polydopamine coating. Cellular functions were studied using fibroblast attachment, cell proliferation, and MTT assays. Fibroblasts were grown on functionalized and pristine CoCr substrates, and the efficacy of BMP-7 peptide on anti-fibrosis was analyzed via gene expression and protein expression of fibrosis markers ACTA2, Collagen 1A1, and fibronectin. The peptide functionalized substrates showed significant reduction of fibrosis markers expression after 1 week of incubation compared to controls. BMP-7 signaling pathway activation was shown by the presence of phosphorylation of Smad1/5/8. These findings may contribute to the improvement of CoCr implants in orthopedic surgery applications.

Covalently grafted BMP-7 peptide to reduce macrophage/monocyte activity: an in vitro study on cobalt chromium alloy.

Cobalt chromium (CoCr) alloy is widely used in orthopedic implants but its functional longevity is susceptible to inflammation related complications. Reduction of the development of chronic inflammation on the biomaterial surface would enhance direct bone-implant bonding and improve implant survival and long-term results. The BMP-7 peptide was derived from the knuckle epitope of bone morphogenic protein-7 (BMP-7) and was conjugated via a cysteine amino acid at the N-terminus. Mouse RAW 264.7 monocytes/macrophages were seeded on the CoCr substrates and inflammation was induced via lipopolysaccharide (LPS) challenge. The effects of BMP-7 peptide on inflammation were evaluated by measuring the expression of inflammatory markers like toll-like receptor-4 (TLR-4), tumor necrosis factor-α (TNF-α), and monocyte chemotactic protein-1 (MCP-1). ELISA and qPCR assays were used to study the inflammatory signals. BMP-7 signaling pathway activation was shown by the presence of phosphorylation of Smad1/5/8. Utilizing the reactivity of polydopamine films to immobilize BMP-7 peptide onto metal substrates may provide a promising approach for applications in situations where reduction of inflammation around implants would be beneficial in improving surgical outcome, bone healing, and implant integration.

Collagen grafted 3D polycaprolactone scaffolds for enhanced cartilage regeneration.

Current surgical and repair treatments for articular cartilage defects still do not give satisfactory long-term results. Scaffold-based tissue engineering is the subject of much intensive interest. However, one major hurdle is that it is unable to accurately replicate the internal three dimensional (3D) microstructure of cartilage. In this work, a novel electrohydrodynamic printing (E-Jetting) technique was employed to fabricate 3D polycaprolactone (PCL) scaffolds, followed by collagen grafting mediated by polydopamine. Surface topography, chemical composition, and wettability of the scaffolds before and after surface functionalization were characterized. Porcine chondrocytes were seeded within the scaffolds for chondrogenesis evaluation. The results showed that a 3D PCL scaffold with controlled fibre diameter, orientation, and pore size was fabricated by the E-Jetting system. The surface functionalization made the PCL scaffold hydrophilic and favourable for cell attachment. The chondrocytes maintained their healthy phenotypes within the collagen grafted PCL scaffold. The increased production of sulfated glycosaminoglycan and highly expressed collagen type II demonstrated that collagen had a positive role in stimulating chondrogenesis and the collagen grafted PCL scaffold was effective in cartilage regeneration.

In vitro characterizations of mesoporous hydroxyapatite as a controlled release delivery device for VEGF in orthopedic applications.

Following bone implant surgery, prolonged ischemic conditions at the implant site often result in postsurgical complications like failure of osseointegration at the bone-implant interface which can lead to implant failure. Thus, restoration of the vascular supply is paramount to the proper development of the bone. High surface area mesostructured materials have been shown to be attractive candidates for bone regeneration to enhance cell adhesion and cell proliferation. This study uses hydroxyapatite, a naturally occurring mineral in the bone, fabricated to a range of suitable pore sizes, infused with vascular endothelial growth factor (VEGF), to be progressively released to stimulate revascularization. In this study, several characterizations including nitrogen adsorption analysis, Fourier-transformed infrared spectroscopy, X-ray diffraction, field emission scanning electron microscope, and transmission electron microscope were used to evaluate the synthesized mesoporous hydroxyapatite (MHA). The results showed that MHA can gradually release VEGF for enhancing revascularization, which is beneficial for orthopedic applications.

WW Domain Containing Transcription Regulator regulates human conjunctiva epithelial cell proliferation via inhibiting TGFß signaling pathway [corrected].

PURPOSE: To investigate the role of Tafazzin (TAZ) protein in regulating the proliferation of normal human conjunctiva epithelial cells and epithelial cells from pterygium tissue. METHODS: Conjunctiva epithelial cells were cultured in keratinocytes growth medium and treated with transformation growth factor ß (TGFß) to analyze the expression and translocation of TAZ protein by immunostaining and BrdU analysis. Immortalized conjunctiva epithelial cells (NHC) were treated with TGFß, targeting siRNA, TGFß receptor antibody or TGFß receptor inhibitor, to study the involvement of TAZ and TGFß signaling pathway in conjunctiva cell proliferation by cell adhesion assay. Conjunctiva tissues from a normal human eye and an eye with pterygium disease were collected for histological analyses and western blot to evaluate the TAZ protein expression in vivo. RESULTS: TAZ expression was upregulated in mitotic conjunctiva epithelial cells, proliferating conjunctiva epithelial cells, TGFß treated conjunctiva epithelial cells and human pterygium epithelium. TAZ siRNA induced less conjunctiva epithelial cell growth. Moreover, TGFß receptor antibody and TGFß receptor inhibitor rescued this anti-proliferative effect of TAZ siRNA. CONCLUSIONS: TAZ is involved in human conjunctiva epithelial cells proliferation via regulating TGFß signaling pathway.

Orthopaedic implant technology: biomaterials from past to future.

Orthopaedic implant technology is heavily based on the development and use of biomaterials. These are non-living materials (e.g. metals, polymers and ceramics) that are introduced into the human body as constituents of implants that fulfill or replace some important function. Examples would be prosthetic joint replacements and fracture fixation implants. For orthopaedic biomaterials to succeed in their desired functions and outcomes in the body, a number of factors need to be considered. The most obvious mechanical properties of the implants are that they need to suit their intended function, and various classes and types of biomaterials have been developed and characterised for use in different implant components depending on their demands. Less well understood but no less important are the interactions that occur between the constituent biomaterials and the living cells and tissues, both of the human host as well as pathogens such as bacteria. Biomaterials used for orthopaedic applications are generally considered to be biocompatible. However, adverse effects arising from interactions at the implant interface can result in various modes of implant failure, such as aseptic loosening and implant infection. This review paper uses the illustrative example of total hip replacement (which has been called the operation of the century) to highlight key points in the evolution of orthopaedic biomaterials. It will also examine research strategies that seek to address some of the major problems that orthopaedic implant surgery are facing today.

Cobalt chromium alloy with immobilized BMP peptide for enhanced bone growth.

Cobalt chromium (CoCr) alloys are widely used in orthopedic practice, however, lack of integration into the bone for long-term survival often occurs, leading to implant failure. Revision surgery to address such a failure involves increased risks, complications, and costs. Advances to enhancement of bone-implant interactions would improve implant longevity and long-term results. Therefore, we investigated the effects of BMP peptide covalently grafted to CoCr alloy on osteogenesis. The BMP peptide was derived from the knuckle epitope of bone morphogenic protein-2 (BMP-2) and was conjugated via a cysteine amino acid at the N-terminus. X-ray photoelectron spectroscopy and o-phthaldialdehyde were used to verify successful grafting at various stages of surface functionalization. Surface topography was evaluated from the surface profile determined by atomic force microscopy. Osteoblastic cells (MC3T3-E1) were seeded on the substrates, and the effects of BMP peptide on osteogenic differentiation were evaluated by measuring alkaline phosphatase (ALP) activity and calcium mineral deposition. The functionalized surfaces showed a twofold increase in ALP activity after 2 weeks incubation and a fourfold increase in calcium content after 3 weeks incubation compared to the pristine substrate. These findings are potentially useful in the development of improved CoCr implants for use in orthopedic applications.

Vascular endothelial growth factor (VEGF) is expressed during articular cartilage growth and re-expressed in osteoarthritis.

INTRODUCTION: Vascular endothelial growth factor (VEGF) is expressed in osteoarthritic articular cartilage. However, the pattern of VEGF expression throughout the whole life cycle of articular cartilage is not well elucidated. The aim of the study was to investigate the spatiotemporal expression of VEGF and its receptors, vascular endothelial growth factor receptor-1 (VEGFR1) and vascular endothelial growth factor receptor-2 (VEGFR2), in articular cartilage during growth, maturation and degeneration, using the guinea pig model of spontaneous osteoarthritis. MATERIALS AND METHODS: Sections of tibial plateaus aged 2, 6 and 12 months were obtained, representing growing, mature and osteoarthritic cartilage respectively. Expression of VEGF and its receptors was determined by immunohistochemistry and in situ hybridisation. RESULTS: At 2 months, VEGF and its receptors were expressed in chondrocytes within the superficial layer of the articular cartilage. At 6 months, no expression of VEGF and its receptors was noted. In the 12-month-old specimens, VEGF and its receptors were expressed in chondrocytes within articular cartilage that exhibited osteoarthritic changes (medial tibial plateaus), but not in the histologically normal lateral plateaus. CONCLUSION: This spatiotemporal distribution of VEGF and its receptors suggests that VEGF is expressed during articular cartilage growth, becomes quiescent at maturity, and is re-expressed in osteoarthritis.

The effect of VEGF functionalization of titanium on endothelial cells in vitro.

One of the key challenges in bone healing and regeneration is the engineering of an implant with surface properties that can enhance revascularization to meet the metabolic demands of recovery. Successful implant integration into the surrounding tissue is highly dependent on the crucial role of blood supply in driving bone repair and development. Therapeutic application of vascular endothelial growth factor (VEGF) is a promising approach to enhance blood supply and healing through revascularization around an engineered implant in a regulated manner. In this in vitro study, we investigated the effects of immobilized VEGF on titanium alloy substrates coated with thin adherent polydopamine film. X-ray photoelectron spectroscopy (XPS) was used to determine the chemical composition of the surfaces at various stages of surface functionalization to verify the successful deposition of polydopamine and VEGF on the metal surface. Surface topography was evaluated from the surface profile determined by atomic force microscopy (AFM). The functionalized surfaces showed a significant increase in human dermal microvascular endothelial cells (HDMECs) attachment, viability and proliferation compared to the pristine substrate. Furthermore the immobilized VEGF was able to induce the differentiation of human mesenchymal stem cells (hMSCs) into endothelial cells. Therefore utilizing the reactivity of polydopamine films to immobilize VEGF onto metal substrates may provide a promising approach for application in situations where revascularization around implants would be beneficial in improving bone healing and implant integration.

Surface functionalization of titanium with carboxymethyl chitosan and immobilized bone morphogenetic protein-2 for enhanced osseointegration.

Orthopedic implant failure has been attributed mainly to loosening of the implant from host bone, which may be due to poor bonding of the implant material to bone tissue, as well as to bacterial infection. One promising strategy to enhance tissue integration is to develop a selective biointeractive surface that simultaneously enhances bone cell function while decreasing bacterial adhesion. In this in vitro study, the surfaces of titanium alloy substrates were functionalized by first covalently grafting carboxymethyl chitosan (CMCS), followed by the conjugation of bone morphogenetic protein-2 (BMP-2) to the CMCS-grafted surface. Bacterial adhesion on the substrates was assayed with Staphylococcus aureus and Staphylococcus epidermidis . Cell functions were investigated using osteoblasts and human bone marrow-derived mesenchymal stem cells. The results showed that bacterial adhesion on both the CMCS and CMCS-BMP-2 functionalized surfaces was significantly reduced compared to that on the pristine substrates. In addition, the CMCS-BMP-2 modified substrates significantly promoted attachment, alkaline phosphatase activity, and calcium mineral deposition of both osteoblast and human bone marrow-derived mesenchymal stem cells. The achievement of the dual functions of bacterial adhesion reduction and cell function promotion by the CMCS-BMP-2 modified titanium substrates illustrates the good potential of such surfaces for enhancement of tissue integration and implant longevity.

Poly (lactic-co-glycolic acid) as a controlled release delivery device.

Poly (lactic-co-glycolic acid) (PLGA) is a biodegradable polymer used to make resorbable sutures, and is also used in other applications in tissue engineering. Being an artificial polymer, its degradation rate can be tailored to suit its application. It can be easily moulded into structures with suitable mechanical strength and degrades into relatively harmless products in the body. Its adjustable degradation rate also makes it a potentially excellent controlled release delivery device. However, the functionalization of PLGA with bioactive molecules usually requires extensive chemical modification. Chemical modification may compromise the mechanical strength of PLGA and inactivate the bioactive molecules. In this paper, a study is done to investigate the coating of an angiogenic factor on unmodified PLGA suture substrates for the differentiation of human mesenchymal stem cells (hMSC) into endothelial cells (EC). The results show that the method used to anchor vascular endothelial growth factor (VEGF) onto the PLGA surface can enable the gradual release of VEGF from the substrate into solution to induce the differentiation of hMSCs into ECs. Thus, this method can potentially be used to coat PLGA materials like sutures, meshes and scaffolds, rendering them functional as effective controlled release delivery devices for a wide range of bioactive molecules.

Human bone marrow-derived mesenchymal stem cells and osteoblast differentiation on titanium with surface-grafted chitosan and immobilized bone morphogenetic protein-2.

Circulating progenitor cells are known to home to various organs to repair injured tissues or to routinely replace old cells and maintain tissue integrity. Similarly, circulating progenitor bone cells can possibly home to a bone implant, differentiate, and eventually osteointegrate with the prosthesis. Osteointegration of bone cells with the prosthesis can help to reduce the risk of implant failure due to constant movement between bone tissue and implant surface. In this study, we aim to investigate if immobilized bone morphogenetic protein-2 (BMP2) on chitosan-grafted titanium substrate (Ti-CS-BMP2) will enhance bone marrow-derived mesenchymal stem cell (BMMSC) adhesion onto the substrate surface and further induce their differentiation into osteoblasts. The results show that our Ti-CS-BMP2 substrate is able to retain adsorbed BMP2, and is capable of slow release of this growth factor. Despite the lesser number of BMMSCs initially attached onto the Ti-CS-BMP2 substrates and consequently the lower level of cell proliferation, Ti-CS-BMP2 cells had the highest level of ALP activity. RT-PCR results show that Ti-CS-BMP2 cells had a relatively higher level of transcription activity of Runx2, compared with that of bone cell-derived osteoblasts (BC-OB), an indication that the BMMSCs were actively differentiating into osteoblasts. Finally, alizarin red staining reveals the presence of calcium deposits in the differentiated cells. Hence our Ti-CS-BMP2 substrates possess an osteoconductive effect and can possibly be used to fabricate bone implants that can osteointegrate with host bone tissue.