The morbidity of bone fractures and defects is steadily increasing due to changes in the age pyramid. As such, novel biomaterials that are able to promote the healing and regeneration of injured bones are needed to overcome the limitations of auto-, allo-, and xenografts, while providing a ready-to-use product that may help to minimize surgical invasiveness and duration. In this regard, recombinant biomaterials, such as elastin-like recombinamers (ELRs), are very promising as their design can be tailored by genetic engineering, thus allowing scalable production and batch-to-batch consistency, among others. Furthermore, they can self-assemble into physically crosslinked hydrogels above a certain transition temperature, in this case body temperature, but are injectable below this temperature, thereby markedly reducing surgical invasiveness. In this study, we have developed two bioactive hydrogel-forming ELRs, one including the osteogenic and osteoinductive bone morphogenetic protein-2 (BMP-2) and the other the Arg-Gly-Asp (RGD) cell adhesion motif. The combination of these two novel ELRs results in a BMP-2-loaded extracellular matrix-like hydrogel. Moreover, elastase-sensitive domains were included in both ELR molecules, thereby conferring biodegradation as a result of enzymatic cleavage and avoiding the need for scaffold removal after bone regeneration. Both ELRs and their combination showed excellent cytocompatibility, and the culture of cells on RGD-containing ELRs resulted in optimal cell adhesion. In addition, hydrogels based on a mixture of both ELRs were implanted in a pilot study involving a femoral bone injury model in New Zealand white rabbits, showing complete regeneration in six out of seven cases, with the other showing partial closure of the defect. Moreover, bone neoformation was confirmed using different techniques, such as radiography, computed tomography, and histology. This hydrogel system therefore displays significant potential in the regeneration of bone defects, promoting self-regeneration by the surrounding tissue with no involvement of stem cells or osteogenic factors other than BMP-2, which is released in a controlled manner by elastase-mediated cleavage from the ELR backbone.

Recombinant Human Parathyroid Hormone (rhPTH 1-34) administration is an effective treatment to improve bone mass in osteoporosis. The aim of this study was to develop a Tissue Engeenering Tool for bone regeneration. We evaluated the efficacy of a freeze dried rhPTH membrane in calvarial critical size defect (CSD). Forty-four Wistar female rats (body weight 150 ± 50 g) with CSD (5 mm) were divided into four groups: group 1: rhPTH membrane (rhPTHm); group 2: atelocollagen membrane (Cm); group 3: rhPTH and atelocollagen I (CrhPTHm); group 4: without any treatment (CG). All samples were evaluated on the 1st, 3rd, and 6th weeks (weeks) post-surgery by soft X-ray, histological and histometric studies. Soft X-ray results showed a radiolucent image with many irregular radiopaque areas. Histologically, rhPTHm was replaced by reticular bone (7%) since 3rd week, and lamellar bone ossicles (30%) at 6th week. Cm showed bone formation like composite bone type on week 1st, 3rd, and 6th (2%, 44%, and 41%, respectively). With CrhPTHm, bone formation was observed in all periods (2.4%, 48%, and 53%), showing statistical difference with CG in the 3rd and 6th wks (p = 0.03 and 0.01). Our results demonstrated the effectiveness of a new biomaterial called CrhPTHm because its ability to regenerate calvarial CSD. Moreover, the membrane represents a new local intermittent delivery system allowing rhPTH slow release.

Hybrid foam (BG-PVA) with 50 % Bioactive glass (BG) and 50 % polyvinyl alcohol (PVA) was prepared by sol-gel process to produce scaffolds for bone tissue engineering. The pore structure of hydrated foams was evaluated by 3-D confocal microscopy, confirming 70% porosity and interconnected macroporous network. In this study, we assessed the putative advantage of coating with osteostatin pentapeptide into BG-PVA hybrid scaffolds to improve their bioactivity. In vitro cell culture experiments were performed using mouse pre-osteoblastic MC3T3-E1 cell line. The exposure to osteostatin loaded-BG-PVA scaffolds increase cell proliferation in contrast with the unloaded scaffolds. An in vivo study was selected to implant BG-PVA scaffolds, non-coated (Group A) or coated (Group B) with osteostatin into non critical bone defect at rabbit femur. Both groups showed new compact bone formation on implant surface, with lamellae disposed around a haversian canal forming osteons-like structure. We observed signs of inflammation around the implanted unloaded scaffold at one month, but resolved at 3 months. This early inflammation did not occur in Group B; supporting the notion that osteostatin may act as anti-inflammatory inhibitor. On the other hand, Group B showed increased bone formation, as depicted by many new trabeculae partly mineralized in the implant regenerating area, incipient at 1 month and more evident at 3 months after implantation. PVA/BG hybrid scaffolds present a porous structure suitable to support osteoblast proliferation and differentiation. Our in vitro and in vivo findings indicate that osteostatin coating improves the osteogenic features of these scaffolds

Bone tissue engineering aims to use biodegrade able scaffolds to replace damaged tissue. This scaffold must be gradually degraded and replaced by tissue as similar as possible to the original one. In this work a hybrid porous scaffold containing chitosan, polyvinyl alcohol and bioactive glass was successfully obtained and subsequently characterized by scanning electron microscopy. The scaffold presented satisfactory pore size range and open interconnected pores, which are essential for tissue ingrowth. A cytotoxicity assay showed that this biomaterial allows adequate cell viability, so that it was considered suitable for an in vivo experiment. Promising results were obtained with the implant of the scaffold in an experimental model of a New Zealand rabbit femur bone lesion. Clinical and biochemical parameters measured such as complete blood count, total serum proteins, albumin, alanine aminotransferase and aspartate aminotransferase were similar between animals in the control group at all time periods studied. Histological and histometric studies showed that the scaffold was coated with a cement-like substance, exhibiting many areas of mineralized structures. Very few osteocyte-like cells or lining-like cells were found inside the amorphous mineralized deposit. In vivo results allow us to consider this scaffold as a promising biomaterial to be applied in bone tissue engineering.

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Bone allografts are commonly used for bone regeneration. The aim of this study was evaluate the efficacy of a freeze dried bone matrix (FDBM) in critical size defect (CZD) rat calvaria. Eighteen Wistar female rats (body weight 150 ± 50 g) with CZD (5mm) were divided in two groups: group 1, using freeze dried bone matrix; and group 2, only with coagulum. All samples were evaluated on the 1st, 3rd and 6th weeks postsurgery by soft X-ray, histological and histometric studies. Soft X-ray results showed a radiolucent image with many irregular radiopaque areas. Histologically, bone regeneration was initiated from the 3rd week, when a thin layer of new woven bone could be seen adjacent to the matrix. At the 6th week, lamellar bone covered over half (61.8 %) of the defect area. The lack of FDBM resorption allowed its incorporation to the new regenerated bone. This behavior is important in circumstances where it is necessary not only to stimulate bone regeneration but also increase the volume in affected areas, such as during the placement of dental implants. The results obtained in this research are encouraging for the use of freeze dried bone matrix as a bone graft material.

In order to evaluate the biocompatibility and mineral repair capacity, mineral trioxide aggregate (MTA), portland cement normal setting (PCn) and fast setting (PCf) and calcium hydroxide-based paste (Calen) filled silicone tube were implanted into the dorsal subcutaneous connective tissues of 25 Wistar rats. Animals were euthanized at 24, 72 hours, 7, 15 and 30 days. Implants with surrounding tissues were fixed with 10% buffer formaldehyde and processed for histological routine techniques. Slides (5 µm serial cuts) were stained with H&E and Von Kossa stains for morphological, qualitative and quantitative analysis by light microscopy. Calen showed severe and moderate inflammatory response and granulomatous reaction with psammoma body-like formation. PCn and MTA have similar behavior, with mild inflammatory reaction from 8% and 4%, respectively. Even though, PCn and MTA demonstrated analogous biological reaction, MTA developing thick artificial mineral precipitation (p = 0,007). All sealers demonstrated a similar inflammatory response at all time periods studied (p = 0.678).

Intermittent Recombinant Human Parathyroid Hormone (rhPTH 1-34) is an effective treatment for improving bone mass in patients with osteoporosis; however, its effects on bone regeneration are still unclear. The objective of this study was to evaluate the potential toxicity systemic rhPTH, as well as its ability to regenerate critical-sized defects (CZD) in bone. We used 43 female Wistar rats (body weight, 150 ± 50 g). Critical-sized bone defects in rat calvaria received vehicle alone (Control Group, CG) or daily rhPTH (20 g/ Kg/day) by subcutaneous injection (Experimental Group, EG). We evaluated bone healing obtained at the 1st, 3rd, and 6th wks post-surgery by biochemical, soft x-ray, histological, and morphometric studies. In the EG, at the 1st and 3rd wks, many areas of focal osteoblast hyperplasia were found on parietal bone. At the 3rd wk, woven and/or lamellar bone, in an organized interconnected trabecular network, showed disrupted mineralization. At the 6th wk, looped bone was found to have formed patterns on parietal bone. New bone formed in the EG showed significant statistical differences (p = 0.023) at the 6th wk. Systemic rhPTH at the dose of 20 g/Kg/ day was able to stimulate bone formation on rat CZD. Also, pre-existing and new bone showed non-proliferative forms of bone hyperostosis (increased non-neoplastic bone).