BackgroundThe injection of mesenchymal stem cells (MSCs) directly into the bone of osteoporotic (OP) patients for rapid recovery has been studied worldwide. Scaffolds associated with MSCs are used to maintain and avoid cell loss after application. A unique heterologous fibrin sealant (HFS) derived from snake venom was evaluated for the cytotoxicity of its main components and as a three-dimensional biological scaffold for MSCs to repair a critical femur defect in osteoporotic rats.MethodsThe cytotoxicity of HFS was assessed using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) assay and transmission electron microscopy. The cells were cultured, characterized by flow cytometry and differentiated into the osteogenic lineage. Two-month-old rats underwent ovariectomy to induce OP. After 3 months, a 5 mm critical bone defect was made in the distal end of the rat femurs and filled with HFS; HFS + MSCs; and HFS + MSCs D (differentiated into the osteogenic lineage) to evaluate the effects. An injury control group (injury and no treatment) and blank control group (no injury and no treatment) were also included. The animals were observed at days 14 and 28 by microtomographic (micro-CT) analyses, histologic and biochemical analysis, as well as scanning electron microscopy.ResultsThe results revealed that one of the compounds of HFS, the thrombin-like enzyme extracted from snake venom, had no cytotoxic effects on the MSCs. OP was successfully induced, as demonstrated by the significant differences in the levels of 17β-estradiol, Micro-CT analyses and alkaline phosphatase between the ovariectomized (OVX) and non-ovariectomized (NOVX) groups. The histological data revealed that at 14 days after surgery in both the OVX and NOVX animals, the HFS + CTMs and HFS + CTMsD showed a higher formation of bone cells at the site in relation to the control group (without treatment). Collagen formation was evidenced through bone neoformation in all treated and control groups. No morphological differences in the femurs of the NOVX and OVX animals were observed after the surgical procedure. Scanning electron microscopy (SEM) confirmed the histological analysis.ConclusionsThe new HFS composed of two non-toxic components for MSCs showed capacity to promote the recovery of the bone lesions in OVX and NOVX animals at 14 days after surgery. In addition, the HFS enabled the differentiation of MSCs into MSCs D in the group treated with HFS + MSCs. Using the MSCs and/or MSCs D together with this biopharmaceutical could potentially enable significant advances in the treatment of osteoporotic fractures. Future clinical trials will be necessary to confirm these results.

Crotalus durissus terrificus (Cdt) venom major components comprise crotoxin, crotamine, gyroxin and convulxin. Crotamine exerts a myotoxic action, among others, but its expression varies even amid snakes from the same region. Biochemical, enzymatic and pharmacological variations of venoms may be associated with the geography, climate, gender, age, and diet, as well as captivity time and venom extraction intervals. The present study aimed to characterize the Cdt venom from the Botucatu region, (SP, Brazil), by assessing its biochemical, pharmacological and enzymatic properties. Venoms from newly captured snakes and already-captured animals were characterized comparatively to verify the sexual, environmental (length of captivity) and ontogenetic variations that could influence the venom composition. Protein concentration, SDS-PAGE and RP-HPLC were performed and the coagulant, toxic (LD50) and crotamine activities were assayed. Individual SDS-PAGE analyses (315 samples) were performed and the biological activities of the venom of 60 adults (captive and newly captured males and females) and 18 newborns were compared with the Brazilian Reference Venom. Crotamine was found in 39.7% (125/315) of the samples, as determined by SDS-PAGE and RP-HPLC. Protein concentration differed significantly between adults (75%) and newborns (60%). RP-HPLC and SDS-PAGE analyses showed highly variable protein concentration and copious crotoxin isoforms; however, the LD50 values decreased during the captivity time. Cdt venom biological activities were similar among adult groups, but diminished during the captivity period. The current findings demonstrate that venoms vary significantly in terms activity and protein concentration, despite originating from the same specie and region.

Fibrin scaffold fits as a provisional platform promoting cell migration and proliferation, angiogenesis, connective tissue formation and growth factors stimulation. We evaluated a unique heterologous fibrin biopolymer as scaffold to mesenchymal stem cells (MSCs) to treat a critical-size bone defect. Femurs of 27 rats were treated with fibrin biopolymer (FBP); FBP + MSCs; and FBP + MSC differentiated in bone lineage (MSC-D). Bone repair was evaluated 03, 21 and 42 days later by radiographic, histological and scanning electron microscopy (SEM) imaging. The FBP + MSC-D association was the most effective treatment, since newly formed Bone was more abundant and early matured in just 21 days. We concluded that FBP is an excellent scaffold for MSCs and also use of differentiated cells should be encouraged in regenerative therapy researches. The FBP ability to maintain viable MSCs at Bone defect site has modified inflammatory environment and accelerating their regeneration.

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