| Background:Soft tissue defect, which results from traumatic injury, tumor resections, congenital defects or aging, often leads to poor cosmesis, affects the emotional well-being of patients, and even impaired function. While these treatments using autograft or allograft materials provide a reasonable degree of clinical success, they have significant drawbacks, including donor-site morbidity, unpredictable outcomes due to graft resorption over time, and allergic reactions. Therefore, there is a strong clinical need for soft tissue substitutes.Injectable fillers have been widely performed as soft tissue substrates in recent years because they are operated in a minimally invasive manner and can easily adapt to complex defects. A number of injectable commercial products are available in the market. However, those materials solely act as fillers and often face limited longevity due to rapid resorption and the risk of potential adverse reactions. A promising approach to solve this problem is the adoption of tissue engineering principle and the use of injectable biodegradable materials, such as collagen, gelatin, fibrin, alginate, silk, and hyaluronic acid, to guide the formation of new soft tissues. Those natural injectable biomaterials are often presented in the form of hydrogels through physical or chemical crosslinking after implanted into the body, and have the advantages of excellent biocompatibility, degradability, and intrinsic cellular interaction. However, the bioengineered grafts using injectable biomaterials alone often had inadequate neovascularization, leading to necrosis or volume reduction after implantation. Vascularization is critical to the survival of engineered adipose tissue. In fact, insufficient vasculature has long been considered as one of the major barriers of regenerating large soft tissues for clinical application.Several approaches have been reported to induce angiogenesis of adipose tissue. For example, co-cultivating human adipose tissue-derived mesenchymal stem cells with endothelial cells was tested to engineer vascularized soft tissues. From a clinical point of view, however, delivery of angiogenic growth factors, such as vascular endothelial growth factor(VEGF), and fibroblast growth factor 2(FGF-2), is a simple and cost-effective strategy for vascularizing soft tissue graft. Adipose tissues extract(ATE), which contains numerous angiogenic factors, was encapsulated in hyaluronan(HA) hydrogel and showed the strong induction in angiogenesis and adipogenesis both in vitro and in vivo. However, the ATE was simply mixed with the injectable HA, and spatial-temporal delivery of the extract from the implant was not well controlled. Collagen hydrogels containing FGF-2 encapsulated in gelatin microspheres was reported to better control FGF-2 delivery and facilitate the development of vascularized adipose tissue. However, how to improve the mechanical property of the collagen gel in order to better support the structure of the newly generated soft tissue has never been solved. Overall, it is desirable to develop new injectable scaffolding system with controlled angiogenic growth factors delivery for soft tissue regeneration.Objective:To develop a tunable injectable biomimetic hydrogel system for rapid angiogenesis, obsreve the revascularization effect of this injectable hydrogel with vascular endothelial growth factor in vivo, and to evaluate its potential as an injectable scaffold for soft tissue regeneration and a carrier for controlled drug delivery.Methods: First, we developed a new approach to incorporate both heparin and tyramine onto gelatin chains, when this gelatin derivative solution was mixed with hydrogen peroxide(H2O2) and horseradish peroxidase(HRP), an enzymatic catalytic reaction takes places and forms an injectable hydrogel in situ. Fourier transform infrared spectroscopy(FTIR), quantification of free amino groups in G/T and G/T/H,and the heparin content measurement, confirming the success of coupling the heparin to the G/T. Then, we test the mechanical properties of this hydrogel, such as gelation time, mechanical strength and degradation in vitro, by changing the concentrations of HRP, H2O2 and G/T/H. BSA and VEGF were incorporated into the G/T and the G/T/H hydrogels and were analysed using BCA method and an ELISA assay, to evaluate its controlled release ability. In order to characterize the effect of VEGF incorporated hydrogel on rapid angiogenesis and tissue regeneration, we first established a cell scratch experimental model to evaluate in vivo bioactivity of the VEGF released from the hydrogels, and then, a chicken chorioallantoic membrane(CAM) assay and animal experiments were performed to further evaluate its rapid angiogenesis effect, by general observation, histopathology and Immunohistochemistry.Results:1. The free amino groups in gelatin chains of G/T was similar to the value of original gelatin(P>0.05), indicating that most of the amino groups in the gelatin chains of the G/T remains intact. In addition, the value of free amino groups in the G/T/H was 50% less than that in the G/T, confirming the success of coupling the heparin to the G/T.2. The gelation time increased with the H2O2 concentration under the same G/T/H and HRP concentration. Under the same G/T/H and H2O2 condition, higher HRP concentration led to shorter time to form a gel. The gelation time also decreased with higher G/T/H concentration.3. The storage modulus of the G/T/H increased with the concentration of H2O2, and reached to the highest value when the H2O2 concentration was 5 mM. Further increasing the concentration of H2O2 led to the decrease of the mechanical strength.4. Low concentration gels degraded faster than the high concentration gels. As the H2O2 increased from low concentration to 5 mM, the degradation rate of the G/T/H decreased, and further increasing H2O2 concentration led to faster G/T/H degradation.5. Both G/T/H and G/T revealed a biphasic release profile, that is, an initial burst of VEGF followed by a gradual and sustained release. However, the burst release of VEGF in the G/T/H group was significantly lower than in the G/T.6. Both the cell number in the scratched region and the average cell migration distance in the control group were significantly shorter than those in the experimental groups, but had no significant difference between experimental groups and VEGF standard group, indicating that the released VEGF(up to 4 weeks) from the G/T/H gels retained its high bioactivity.7. After 5 days of incubation, the control groups showed a normal CAM vessel network, and the growth process of the blood vessels was of the random state. In contrast, more blood vessels grew radially towards the VEGF-containing hydrogels than the control, Quantitative analysis confirmed that the number of blood vessels surrounding the VEGF-containing G/T(30±6) and G/T/H(42±4) gels were significantly higher than the control group(18±3, P<0.05). Additionally, the number of new blood vessels in the G/T/H+VEGF group also showed a significant increase compared to the G/T+VEGF group(P<0.05).8. After subcutaneously injected hydrogel into the dorsal side of mice for 2 weeks, the blood vessel was scarcely found on the surface of the G/T hydrogel. For the G/T/H specimens, more blood vessels were observed surrounding the hydrogel and a few of them extended inside the gel. H&E staining shows that the G/T group had a minor cell infiltration along the edge of the gel, whereas the other three groups all showed a moderate cell infiltration. The blood vessel distribution in the gels was similar to that of the cell infiltration.Conclusions:This study presents a tunable injectable biomimetic hydrogel for rapid angiogenesis. The properties of this injectable hydrogel, such as gelation time, mechanical strength and degradation rate, could be controlled by altering the HRP, H2O2 and the polymer concentration, and the amount of modified heparin can be mediated by its reaction content. The in vitro and in vivo data demonstrated that modification of the G/T scaffold with heparin not only controlled release VEGF in a sustained manner, but also retained its activity for a longer time. In addition, without the incorporation of VEGF, this G/T/H hydrogel still have the ability to induce angiogenesis to a certain degree. In conclusion, the G/T/H hydrogel has great potential for use as an injectable scaffold for tissue regenerative medicine and as a drug carrier for controlled drug delivery systems. |
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