1. Introduction

Guided bone regeneration (GBR) in the oral cavity is a procedure that attempts to restore bone tissue in a specific area, by the use of a cell occlusive membrane. To reach this goal, the cellular proliferation originating from the adjacent bone onto a scaffold support must have a higher proliferative rate than fibrogenesis coming from surrounding connective and soft tissues.^[1] Soft tissue regeneration and remodeling is fundamental, especially in the case of exposed roots and gingival recession, and to increase peri-implant mucosal tissues. The PEG membrane fulfils the main criteria for a resorbable GBR membrane having a cell-occlusive function,^[2] slow reabsorption time,^[3] and the ability to allow bone regeneration.^[4] PEG membrane thus lead to similar amounts of bone regeneration as a polytetrafluoroethylene (ePTFE) membrane.^[5] PEG hydrogel shows unique biochemical properties that make it well suited for use as a carrier for other bioactive materials, as for RGD,^[3] enamel matrix derivative (EMD), and nanobioglasses (NBG). RGD is a tripeptide (Arg-Gly-Asp) that by binding with integrins (transmembrane receptors that bind cells to the extracellular matrix) enhances bone regeneration.^[6] EMD is an extract of enamel matrix and contains amelogenins of various molecular weights, involved in the formation of enamel and periodontal formation during tooth development. EMD stimulates the regeneration of the periodontium, reduces crevicular depth, and enhances the formation of a new epithelial attachment in cases of periodontal bone defects.^[7] Recent studies^[8] have shown that EMD, and more specifically amelogenin (very likely the 28.9-kDa protein), stimulates angiogenesis. Angiogenesis is a fine-tuned mechanism intimately linked to a wide variety of both normal and pathological processes. Inadequate or inappropriate bone vascularity is associated with decreased bone formation and repair.^[9] PEG hydrogel can also be conjugated with NBGs, a group of surface reactive glass–ceramic biomaterials able to stimulate bone regeneration and form a bond with the bone itself. Moreover, NBG display an antimicrobial effect due to the release of alkaline species.^[10] Considering these observations, this study was designed to investigate the angiogenic response elicited by PEG hydrogel alone or preconditioned with EMD or NBG in skin wounds produced in experimental animals.

2. Materials and Methods

2.5. Blood flow evaluation

For blood flow evaluation, Laser Doppler tests were performed at baseline, 1, 2, 4, 8, and 16 weeks using a perfusion imager (PIM 3.0 Perimed/Periscan, Sweden). Based on the Doppler principle, the instrument scans the tissue with a laser beam and collects back-scattered light without tissue contact. Color-coded images are generated showing the spatial distribution of the tissue perfusion (red=high perfusion, yellow=moderate, green=low, blue=very low perfusion).

The generated images together with the graph representing flow variation were used for the subsequent analysis. A region of interest (ROI) was selected around the same pocket at all time points in all time points (basal, 1-2-4-8-16 weeks) and the perfusion of the empty pocket was set as reference. To select the ROI, the area underneath the suture thread (for the first time points) or the scar (for the last three time points) was used in the recorded photos (Fig. 1A).

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FIG. 1.

(A) Representative Laser Doppler software appearance during the evaluations. (B) Results from Laser Doppler analysis at 1, 2, 4, 8, and 16 weeks following biomaterial insertion. Bars in the graph represent the mean of the results obtained from seven animals. ***p<0.0001 versus other PEG preparations, **p<0.001 versus PEG, #p<0.005 versus empty pouch. Color images available online at www.liebertpub.com/tea

Data generated by the PIM 3 software were then plotted in the GraphPad Prism 5 software and analyzed.

3. Results

3.2. Histology results

At 16 weeks, PEG+EMD appeared to be the biomaterial, which was most integrated in the surrounding tissues, in agreement with the highest blood flow observed by Laser Doppler. Between the first and the last week, no further differences were observed in the analyzed samples (Fig. 2). No inflammatory reaction was observed in all the evaluated samples at any time point, as evidenced by the negative MPO staining (Fig. 3).

FIG. 2.

Representative histological skin appearance after 16 weeks from biomaterial insertion. (A) empty, (B) PEG, (C) PEG-EMD, and (D) PEG-NBG. The arrows indicate nonhomogeneous areas where likely the biomaterial was integrated into the tissue. Color images available online at www.liebertpub.com/tea

FIG. 3. Representative MPO staining in the skin after 1 week from biomaterial insertion. (A) empty, (B) PEG, (C) PEG-EMD, (D) PEG-NBG. The arrows in (A) indicates slight positive reaction around the vessel wall. Color images available online at www.liebertpub.com/tea

The staining with CD-31 (Fig. 4A–D) showed the presence of few and slightly marked new vessels from week 2 up to week 8 when angiogenesis and remodeling occurred following surgery in the empty pocket. PEG alone was able to improve the number of small-caliber vessels (representing active angiogenesis), especially at week 4. Preconditioning of PEG with EMD showed the most impressive results with the largest increase in positive staining as early as week 1 and persistence throughout week 8. PEG combined with NBG also showed a significant increase in angiogenesis starting from week 4 to 8 (Fig. 4E).

FIG. 4.

A-D Representative CD31 staining in the skin after different weeks from biomaterial insertion. (A) PEG-EMD at 1 week, (B) PEG-EMD at 2 weeks, (C) PEG-NBG at 4 weeks, and (D) PEG-NBG at 8 weeks. The arrows indicate positive small-caliber vessels. (E) Microvascular density evaluated at each time point in several groups. ***p<0.0001 versus empty and other PEG preparations, **p<0.05 versus empty pouch. Color images available online at www.liebertpub.com/tea

4. Discussion

In recent years, several biocompatible materials belonging to different categories have been successfully used for GBR procedures, e.g. nonresorbable (ePTFE, titanium mesh), and resorbable materials (dura mater, polylactic acid, polyglycolic acid, and polyurethane). To be used in GBR procedures, each material must meet specific criteria such as biocompatibility, tissue integration, cell occlusivity, nutrient permeability, degradation kinetics, and ease of use during surgery.^[3] Nonresorbable membranes require a second surgery to be retrieved and, in case of premature exposure, there is a higher chance of bacterial colonization.

Consequently, current investigations are focused on resorbable devices, especially on type I and type III porcine or bovine collagen membranes.^[12] Collagen meets certain important requirements of a barrier for GBR procedures, including early wound stabilization, chemotactic properties to attract fibroblast, and semipermeability, but its degradation could be accelerated by polymorphonuclear leucocytes, macrophages, and periodontal bacterial pathogens resulting in early membrane collapse. The heterologous nature of collagen by-products can be related to adverse reactions, thus compromising tissue integration. Moreover, all prefabricated devices must be adapted to the individual defect, therefore compromising the handling in sites that are difficult to reach.

To overcome these drawbacks, recent experimental studies, conducted on both animal and human models, have investigated the efficacy of a newly developed synthetic hydrogel made of polyethylene glycol (PEG) to be used in bone regeneration therapy.

The purpose of this study was to analyze the role of a proprietary PEG gel formulation as a synthetic barrier, able to stimulate the angiogenic response in skin wounds, in an experimental animal model. In this study, the hydrogel was studied alone or preconditioned by adsorption of specific amelogenins or nanobioglass particles, to evaluate whether these components might modify the ability of the hydrogel to increase angiogenesis. The animal model and the surgical procedure were conducted in accordance with the previous studies proposed by Herten et al.³

To the best of our knowledge, this is the first published report of the use of Laser Doppler analysis to study perfusion of superficial skin layers following the insertion of biomaterials commonly used for oral tissue regeneration. This technique allowed creation of a perfusion map of blood flow variations at the surgical site following different times after the surgical procedure. The PEG gel formulation was shown to be a valid resorbable barrier membrane with evidence of hydrogel remnants at 16 weeks, but no evidence of inflammatory reaction or tissue necrosis.

Laser Doppler analysis highlighted statistically relevant modifications at 1 week for EMD and 2 weeks for nanobioglass. Histological analysis confirmed the angiogenetic response to both EMD and nanobioglass. EMD, whose angiogenetic potential was already reported,^[8] is able to raise a statistically significant response after 1, 2, and 8 weeks, but not after 4 weeks. One possible explanation for this pattern may be the decreasing progression of angiogenesis, which reaches a plateau at the fourth week and does not further progress until the eighth week.

In contrast, the nanobioglass reaches the highest point of vascular formation after 4 weeks, with a slower response. One possible explanation is that EMD, as an active biomolecule, is able to cause an angiogenetic burst in the early phases of wound healing, while the nanobioglass, as a scaffold, shows a slower, less intense but more steady action. The MPO assay suggests that the EMD angiogenetic stimulus is a direct action and not a consequence of an inflammatory reaction.^[3]

Angiogenesis plays a fundamental role in the wound healing process, so wherever the vascular conditions are lacking at the surgical site, consistent neoangiogenetic stimuli could enhance the healing of the wound.^[13][14][15]The results described here suggest that PEG is able to interact with the host tissue without any inflammatory reaction, regardless of the previous preconditioning of the material with other substances. In addition, we have demonstrated an increase in blood flow and angiogenesis in the area where PEG was inserted in combination with EMD, which was able to further improve PEG integration in the host tissues as well as the angiogenic response for up to 8 weeks.

This study shows how PEG, besides being a valid barrier membrane, can act as a carrier for different bioactive molecules, likely mediating their release. The substances used in our experiments, especially the EMD, appeared to improve neoangiogenesis, possibly enhancing the healing course in all places that a strong vascular support is needed, as in the GBR.

The use of biomaterials has dramatically improved tissue regeneration procedures and patient outcomes, however, several conditions may alter this process. In particular, altered blood flow supply, reduced production, and release of growth factors and their related molecules could negatively affect the healing process, as it occurs in diabetes.^[16] These results provide new insights on the regenerative efficacy of biomaterials, exploring the angiogenic and the tissue remodeling response, making these biomaterials useful for a possible immediate use in the clinical setting. If confirmed, these data could be extremely important to improve the poor outcome, due to other preexisting conditions (i.e., smoking habit or diabetes), in regenerative procedures.

Future investigations could be directed toward a combination of these three materials compounded in a single formulation, thus combining all advantages and verifying the interactions between the components.