A Novel Drastic Peptide Genetically Adapted to Biomimetic Scaffolds "Delivers" Osteogenic Signals to Human Mesenchymal Stem Cells.

This work describes the design, preparation, and deep investigation of "intelligent nanobiomaterials" that fulfill the safety rules and aim to serve as "signal deliverers" for osteogenesis, harboring a specific peptide that promotes and enhances osteogenesis at the end of their hydrogel fibers. The de novo synthesized protein fibers, besides their mechanical properties owed to their protein constituents from elastin, silk fibroin and mussel-foot adhesive protein-1 as well as to cell-attachment peptides from extracellular matrix glycoproteins, incorporate the Bone Morphogenetic Protein-2 (BMP2) peptide (AISMLYLDEN) that, according to our studies, serves as "signal deliverer" for osteogenesis. The osteogenetic capacity of the biomaterial has been evidenced by investigating the osteogenic marker genes ALP, RUNX2, Osteocalcin, COL1A1, BMPR1A, and BMPR2, which were increased drastically in cells cultured on scaffold-BMP2 for 21 days, even in the absence of osteogenesis medium. In addition, the induction of phosphorylation of intracellular Smad-1/5 and Erk-1/2 proteins clearly supported the osteogenetic capacity of the biomaterial.

Chitosan/PEGDA based scaffolds as bioinspired materials to control in vitro angiogenesis.

In the current work, our purpose was based on the assessment of bioactive chitosan (CS)/Poly(ethylene glycol) diacrylate (PEGDA) based scaffolds ability to stimulate in vitro angiogenesis process. The bioactivation of the scaffolds was accomplished by using organic (BMP-2 peptide) and inorganic (hydroxyapatite nanoparticles) cues. In particular, the properties of the materials in terms of biological response promotion on human umbilical vein endothelial cells (HUVECs) were studied by using in vitro angiogenesis tests based on cell growth and proliferation. Furthermore, our interest was to examine the scaffolds capability to modulate two important steps involved in angiogenesis process: migration and tube formation of cells. Our data underlined that bioactive signals on CS/PEGDA scaffolds surface induce a desirable effect on angiogenic response concerning angiogenic marker expression (CD-31) and endothelial tissue formation (tube formation). Taken together, the results emphasized the concept that bioactive CS/PEGDA scaffolds may be novel implants for stimulating neovascularization of tissue-engineered constructs in regenerative medicine field.

Periosteum Mimetic Coating on Structural Bone Allografts via Electrospray Deposition Enhances Repair and Reconstruction of Segmental Defects.

Structural bone allograft transplantation remains one of the common strategies for repair and reconstruction of large bone defects. Due to the loss of periosteum that covers the outer surface of the cortical bone, the healing and incorporation of allografts is extremely slow and limited. To enhance the biological performance of allografts, herein, we report a novel and simple approach for engineering a periosteum mimetic coating on the surface of structural bone allografts via polymer-mediated electrospray deposition. This approach enables the coating on allografts with precisely controlled composition and thickness. In addition, the periosteum mimetic coating can be tailored to achieve desired drug release profiles by making use of an appropriate biodegradable polymer or polymer blend. The efficacy study in a murine segmental femoral bone defect model demonstrates that the allograft coating composed of poly(lactic-co-glycolic acid) and bone morphogenetic protein-2 mimicking peptide significantly improves allograft healing as evidenced by decreased fibrotic tissue formation, increased periosteal bone formation, and enhanced osseointegration. Taken together, this study provides a platform technology for engineering a periosteum mimetic coating which can greatly promote bone allograft healing. This technology could eventually result in an off-the-shelf and multifunctional structural bone allograft for highly effective repair and reconstruction of large segmental bone defects. The technology can also be used to ameliorate the performance of other medical implants by modifying their surfaces.

Bioactivation Routes of Gelatin-Based Scaffolds to Enhance at Nanoscale Level Bone Tissue Regeneration.

The present work is focused on the development of gelatin-based scaffolds crosslinked through carbodiimide reaction and their bioactivation by two different methods: (i) surface modification by inorganic signals represented by hydroxyapatite nanoparticles precipitated on scaffold through biomimetic treatment; (ii) analog of BMP-2 peptide decoration. The results showed the effects of polymer concentration and crosslinking time on the physico-chemical, morphological, and mechanical properties of scaffolds. Furthermore, a comparative study of biological response for both bioactivated structures allowed to evaluate the influence of inorganic and organic cues on cellular behavior in terms of adhesion, proliferation and early osteogenic marker expression. The bioactivation by inorganic cues induced positive cellular response compared to neat scaffolds in terms of increased cell proliferation and early osteogenic differentiation of human mesenchymal stem cell (hMSC), as evidenced by the Alkaline phosphatase (ALP) expression. Similarly BMP-2 peptide decorated scaffolds showed higher values of ALP than biomineralized ones at longer time. The overall results demonstrated that the presence of bioactive signals (either inorganic or organic) at nanoscale level allowed an osteoinductive effect on hMSC in a basal medium, making the modified gelatin scaffolds a promising candidate for bone tissue regeneration.

Fabrication of a biomimetic ZeinPDA nanofibrous scaffold impregnated with BMP-2 peptide conjugated TiO2 nanoparticle for bone tissue engineering.

A biomimetic Zein polydopamine based nanofiber scaffold was fabricated to deliver bone morphogenic protein-2 (BMP-2) peptide conjugated titanium dioxide nanoparticles in a sustained manner for investigating its osteogenic differentiation potential. To prolong its retention time at the target site, BMP-2 peptide has been conjugated to titanium dioxide nanoparticles owing to its high surface to volume ratio. The effect of biochemical cues from BMP-2 peptide and nanotopographical stimulation of electrospun Zein polydopamine nanofiber were examined for its enhanced osteogenic expression of human fetal osteoblast cells. The sustained delivery of bioactive signals, improved cell adhesion, mineralization, and differentiation could be attributed to its highly interconnected nanofibrous matrix with unique material composition. Further, the expression of osteogenic markers revealed that the fabricated nanofibrous scaffold possess better cell-biomaterial interactions. These promising results demonstrate the potential of the composite nanofibrous scaffold as an effective biomaterial substrate for bone regeneration.

3-D mineralized silk fibroin/polycaprolactone composite scaffold modified with polyglutamate conjugated with BMP-2 peptide for bone tissue engineering.

In the field of bone tissue engineering, an ideal three-dimensional (3-D) scaffold should not only structurally mimic the extracellular matrix (ECM) in large tissues but also mechanically support the bone healing process and provide biochemical cues to induce osteogenesis. In this study, we investigated the feasibility of functionalisation of scaffolds by coupling polyglutamate acid conjugated with BMP-2 peptide onto silk fibroin (SF)/polycaprolactone (PCL) (SF/PCL) blend nanofibers. The morphology, composition, and mineralisation, were confirmed by FE-SEM, XRD, and FT-IR spectroscopy. The FE-SEM images revealed that wet-electrospun nanofibrous scaffolds exhibited inter-connected nano/micro-pores at different levels, and a different morphology was observed on the 3-D SF/PCL scaffold after mineralisation. Furthermore, the binding property and release behaviour of the peptide were investigated on this mineralized structure, and adipose-derived stem cells were seeded on the composite scaffolds to assay their cytocompatibility and osteogenic differentiation capacities. Results suggest that the polyglutamate motif (repetitive glutamate amino acids) exhibited markedly improved binding properties to mineralized nanofibers, and the mineralized 3-D scaffolds with the conjugated with peptide enhances the mRNA expression of osteogenic genes. The sponge-like 3-D nanofibrous scaffold mechanically and biochemically mimics the regenerative process for applications in bone tissue engineering, including the regeneration of calvarial defects.

In vitro BMP-2 peptide release from thiolated chitosan based hydrogel.

Thiolated chitosan based thermo-sensitive hydrogel is a water soluble system and the existing thiol groups are beneficial for the delivery of cysteine-rich peptides. In the present study, a kind of thiolated chitosan, i.e. chitosan-4-thio-butylamidine (CS-TBA) conjugate was characterized and used to prepare CS-TBA/hydroxyapatite (HA)/beta-glycerophosphate disodium (ß-GP) thermo-sensitive hydrogel. The cysteine terminated peptide 24 (P24) containing residues 73-92 of the knuckle epitope of BMP-2 (N→C: KIPKASSVPTELSAISTLYLSGGC) was synthesized and characterized. The release behavior of P24 from CS-TBA based hydrogel was investigated in vitro. The thiol groups in CS-TBA may react with thiol groups in P24, thus decreases the P24 release rate and maintains the peptide release for a longer time compared with unmodified chitosan based hydrogel. Moreover, the bioactivity of P24 is preserved during release process. These results indicate that P24 loaded CS-TBA based thermosensitive hydrogel is a potential material for minimally invasive surgery of bone repair.

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.