3D printed mesh reinforcements enhance the mechanical properties of electrospun scaffolds.

BACKGROUND: There is substantial interest in electrospun scaffolds as substrates for tissue regeneration and repair due to their fibrous, extracellular matrix-like composition with interconnected porosity, cost-effective production, and scalability. However, a common limitation of these scaffolds is their inherently low mechanical strength and stiffness, restricting their use in some clinical applications. In this study we developed a novel technique for 3D printing a mesh reinforcement on electrospun scaffolds to improve their mechanical properties. METHODS: A poly (lactic acid) (PLA) mesh was 3D-printed directly onto electrospun scaffolds composed of a 40:60 ratio of poly(ε-caprolactone) (PCL) to gelatin, respectively. PLA grids were printed onto the electrospun scaffolds with either a 6 mm or 8 mm distance between the struts. Scanning electron microscopy was utilized to determine if the 3D printing process affected the archtitecture of the electrospun scaffold. Tensile testing was used to ascertain mechanical properties (strength, modulus, failure stress, ductility) of both unmodified and reinforced electrospun scaffolds. An in vivo bone graft model was used to assess biocompatibility. Specifically, reinforced scaffolds were used as a membrane cover for bone graft particles implanted into rat calvarial defects, and implant sites were examined histologically. RESULTS: We determined that the tensile strength and elastic modulus were markedly increased, and ductility reduced, by the addition of the PLA meshes to the electrospun scaffolds. Furthermore, the scaffolds maintained their matrix-like structure after being reinforced with the 3D printed PLA. There was no indication at the graft/tissue interface that the reinforced electrospun scaffolds elicited an immune or foreign body response upon implantation into rat cranial defects. CONCLUSION: 3D-printed mesh reinforcements offer a new tool for enhancing the mechanical strength of electrospun scaffolds while preserving the advantageous extracellular matrix-like architecture. The modification of electrospun scaffolds with 3D-printed reinforcements is expected to expand the range of clinical applications for which electrospun materials may be suitable.

Sustained delivery of the angiogenic QK peptide through the use of polyglutamate domains to control peptide release from bone graft materials.

Angiogenesis plays a pivotal role in tissue regeneration following bone-grafting procedures; however, nonautogenous graft materials typically lack critical angiogenic growth factors. While much research has focused on modifying grafts with angiogenic factors, controlled delivery of these molecules remains a challenge. The current study describes a method for sustained delivery of an angiogenic peptide from hydroxyapatite (HA), a common alloplast material. Specifically, VEGF-derived "QK" peptides were synthesized with polyglutamate domains containing varying numbers of glutamates. The rate of peptide release from HA inversely correlated with glutamate number, with diglutamate-QK (E2-QK) released first, followed by tetraglutamate-QK (E4-QK), and finally, heptaglutamate-QK (E7-QK). By coating HA with a mixture of these peptides, termed, PGM-QK (polyglutamate-modified mixture), sequential peptide release was achieved, enabling gradient QK delivery. To evaluate bioactivity, HA disks were coated with PGM-QK and then placed in fresh media for 6 days. Media containing the released peptides was collected at varying time intervals and placed on human umbilical vein endothelial cells (HUVECs). Cells were evaluated for activation of angiogenic signaling pathways (ERK and Akt) and cell migration. Results showed that QK peptides were continuously released over the 6-day interval, and maintained their capacity to activate HUVECs. These findings point to a new approach for gradient delivery of an angiogenic stimulus.

Addition of an oligoglutamate domain to bone morphogenic protein 2 confers binding to hydroxyapatite materials and induces osteoblastic signaling.

Nonautologous bone grafts have limited osteoinductive potential and thus there is substantial interest in reconstituting these graft materials with osteogenic factors such as bone morphogenic protein 2 (BMP2). However, one limitation of this approach is that BMP2 is typically weakly bound to the graft, which can lead to side effects associated with BMP2 dissemination. In the current study we added a hydroxyapatite (HA)-binding domain onto BMP2 to increase coupling to the graft surface. A sequence consisting of eight glutamate residues (E8) was inserted into the C-terminus of BMP2, and the recombinant protein (rBMP2-E8) was expressed in E. coli. Compared with rBMP2, rBMP2-E8 displayed markedly enhanced binding to HA disks and was better retained on the disks following exposure to vigorous wash steps. Furthermore, rBMP2-E8 was purified using a heparin column, and evaluated for its capacity to stimulate osteoblastic cell signaling. Treatment of SAOS2 cells with rBMP2-E8 induced SMAD 1/5 activation, confirming that the protein retains activity. Collectively these results suggest that the E8 domain serves as an effective tool for improving rBMP2 coupling to graft materials. The increased retention of rBMP2-E8 on the graft surface is expected to prolong BMP2's osteoinductive activity within the graft site, while simultaneously reducing off-target effects.

The addition of a polyglutamate domain to the angiogenic QK peptide improves peptide coupling to bone graft materials leading to enhanced endothelial cell activation.

Vascularization of bone grafts is vital for graft integration and bone repair, however non-autologous graft sources have limited potential to induce angiogenesis. Accordingly, intensive research has focused on functionalizing non-autologous materials with angiogenic factors. In the current study we evaluated a method for coupling an angiogenic peptide to the surface of two clinically-relevant graft materials, anorganic bovine bone (ABB) and synthetic hydroxyapatite (HA). Specifically, the VEGF-derived "QK" peptide was synthesized with a heptaglutamate (E7) domain, a motif that has strong affinity for calcium phosphate graft materials. Compared with unmodified QK, a 4-6 fold enrichment was observed in the binding of E7-modified QK (E7-QK) to ABB and HA. The E7-QK peptide was then assessed for its capacity to stimulate angiogenic cell behaviors. Human umbilical vein endothelial cells (HUVECs) were treated with solutions of either QK or E7-QK, and it was found that QK and E7-QK elicited equivalent levels of cell migration, tubule formation and activation of the Akt and ERK signaling pathways. These data confirmed that the inherent bioactivity of the QK sequence was not diminished by the addition of the E7 domain. We further verified that the activity of E7-QK was retained following peptide binding to the graft surface. HA disks were coated with QK or E7-QK, and then HUVECs were seeded onto the disks. Consistent with the increased amount of E7-QK bound to HA, relative to QK, markedly greater activation of Akt and ERK 1/2 was observed in cells exposed to the E7-QK-coated disks. Taken together, these results suggest that the E7 domain can be leveraged to concentrate angiogenic peptides on graft materials, facilitating delivery of higher peptide concentrations within the graft site. The ability to endow diverse graft materials with angiogenic potential holds promise for augmenting the regenerative capacity of non-autologous bone grafts.

Taking cues from the extracellular matrix to design bone-mimetic regenerative scaffolds.

There is an ongoing need for effective materials that can replace autologous bone grafts in the clinical treatment of bone injuries and deficiencies. In recent years, research efforts have shifted away from a focus on inert biomaterials to favor scaffolds that mimic the biochemistry and structure of the native bone extracellular matrix (ECM). The expectation is that such scaffolds will integrate with host tissue and actively promote osseous healing. To further enhance the osteoinductivity of bone graft substitutes, ECM-mimetic scaffolds are being engineered with a range of growth factors (GFs). The technologies used to generate GF-modified scaffolds are often inspired by natural processes that regulate the association between endogenous ECMs and GFs. The purpose of this review is to summarize research centered on the development of regenerative scaffolds that replicate the fundamental collagen-hydroxyapatite structure of native bone ECM, and the functionalization of these scaffolds with GFs that stimulate critical events in osteogenesis.