Most clinicians believe that it is preferable to have keratinized gingiva adjacent to teeth to perform oral hygiene easier, resist frenum pull, reduce further recession. ¹ , ² Inadequate keratinized mucosa may be due to high plaque accumulation and gingival inflammation. ³ As a standard and routine technique, free gingival graft (FGG) has been used to increase the zone of keratinized gingiva. ³ , ⁴

Despite favorable results from this technique, some disadvantages guided investigators to seek for better alternatives. ⁴ As a donor, the palatal tissue provides keratinized gingiva, however, the problems still exist in relation to the palatal phenotype, the diversity of color and texture with surrounding mucosa, ⁵ and the bulky appearance which seems to be an esthetic problem in anterior sites. ⁴ , ⁵ , ⁶ Most importantly, the need for a donor site adds to the patient morbidity (e.g., pain and bleeding). ⁴ , ⁵ , ⁶

Acellular dermal matrix (ADM) allograft has been used to cover full-thickness burn wounds. ⁷ Not long ago, ADM has been introduced to increase width of keratinized gingiva (KGW) as an alternative to autogenous palatal graft. ⁴ , ⁶ ADM is made of skin in split-thicknesses with no cellular components (fibroblasts, keratinocytes, vascular endothelium, sebaceous glands, sweat glands, and smooth muscle). ⁸ , ⁹ , ¹⁰ , ¹¹ , ¹² ADM consists of a basement membrane complex and extracellular matrix proteins; ⁶ therefore, it is thought to be weakly immunogenic or nonimmunogenic. Although it is accepted well in human tissues as an allogeneic graft, ⁹ , ¹⁰ , ¹¹ the possible risk of disease transmission, cost of commercially available human ADM, and at the same time limited availability of cadaver skin, limit the use of this material as a dermal replacement. ⁹ It is assumed that ADM from xenogeneic source may solve the above problems, so it will become as a favorite material to use in a broad spectrum of periodontal treatment procedures. ⁹ , ¹³ Recently, the use of xenogeneic ostrich skin as a new and available source for ADM in surgical procedures in skin deficit was considered. ¹³

To the best of our knowledge at best, there are no published studies in dentistry showing the use of this xenogeneic ADM. Thus, for the first time, it was aimed to compare the clinical efficacy, histological structure, histomorphometric properties, and biocompatibility of the ostrich ADM with autogenous FGG, when grafted into alveolar mucosa has led to increase of width of keratinized attached tissue in dogs. Our primary outcomes were to compare the KGW and color match following Ostrich ADM and FGG, while the secondary outcomes were to analyze histological parameters.

Animals and presurgical procedures

Ten mixed breed male dogs, aged 1–1.5, and weighing 15–20 kg were chosen for this split-mouth animal study.

The sample size was calculated on the basis of results achieved from a preliminary sample of 5 dogs, all eligible for the subject of study.

According to the preliminary results, changes on the keratinized gingival thickness of the two treatment methods were 0.24 ± 0.15. Thus, the final necessary sample size according to paired sample size formulation is n = 10.

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Where α <0.01; β <0.05, therefore n = 10

The animals were examined by a veterinarian in Faculty of Veterinary Medicine, Science and Research Branch, Islamic Azad University at Tehran and vaccination was done for the animals. Oral examination was done; there were no buccal lesions in the mouth. Animals were kept in individual metal cages, provided commercial pet pad in the surgery department, science, and research center. The dogs were anestrous and kept at 25°C at normal day/light cycle. All animals provided commercial pet food and tap water. The animals were under suitable regimen for 2 weeks, during this period the dogs' teeth were cleaned carefully to control the gingival inflammation. Then, animals randomly allocated into experimental groups. The protocol was approved by the Animal Ethics Committee (no: 85-23). All experimental procedures were carried out in accordance to the Guide for the Care and Use of Laboratory Animals to Investigate Experimental Pain in Animals. ¹⁴ Animal handling and experimental procedures were in compliance with ARRIVE guidelines [Flowchart 1].[INLINE:2]

Preparation of acellular dermal matrix

Fresh skin was obtained from normal and young (1–1/5 year) male ostrich abdomen under aseptic condition. Cleaning and excision of subdermal fatty tissues were done in clean room Class B (Tissue Bank research center). The ostrich skin was then washed with phosphate-buffered saline (PBS) (PBS-Gibco-UK) and kept in Antibacterial media (Mixed of Gram positive and negative antibiotic) for 24 h. The samples were washed out 5 times entirely by Distilled water (DW) and soaked in Triton X-100 (Sigma-UK) at 4°C temperature for 24 h. The processed skins were washed again in DW and kept in sodium dodecyl sulfate (SDS) (SDS, Merck, Germany) for the next 15 h in 4°C temperature. On the next step, the samples were washed in DW and kept in SDS for another 10 h. Finally, the samples were washed in PBS. All solutions which were used in ADM preparation were filter-sterilized, and the procedures were performed aseptically. To prevent microbial growth, Sodium Azide, (0.02 gr/100 ml) (Merck, Germany) was present at all times in the extracted solutions. ¹³ At the end, big samples were divided to 40 equal square shape derms (20 mm × 20 mm). The samples were washed for 10 times and freeze derided by lyophilized Zirbus-Germany machine. The samples were packed in double sealed bristles and sterilized by 25 k gray Gamma irradiate (Atomic Research Center).

Scanning electron microscope evaluation

A random freeze dried sample selected for scanning electron microscope (SEM) evaluation Figure 1. A sample dimension about 3 mm × 3 mm was cut and attached on SEM disc then coated by Gold. The disc was placed in SEM chamber (JEOL 1000, USA). SEM result showed rough surface without cells and ostrich feather. Figure 1

Scanning electron microscope view of a freeze dried sample of ostrich skin.

Pre- and post-surgical measurements were performed by a periodontist not involved in the surgeries. Measurements were made at the nearest 0.5 mm using a Marquis Color coded periodontal probe (Hufridy, USA). The mean score for each variable was calculated, and the results were used in the statistical analysis.

The mean KGW before and 1, 3, 6 months after surgery in the study groups is shown in Table 1.{Table 1}

The results of the study showed that the mean KGW within the study groups, after surgery and in interval examinations, had been increased significantly compare to the baseline (P = 0.001 in both groups), however, there was no statistically significant difference between the groups (P > 0.05).

After 6 months from the initiation of treatment, the mean KGWs in the control and test group were 8.15 ± 0.27 and 8.07 ± 0.24 mm, respectively. At this time, control and test groups showed 4.65 ± 0.50 and 4.8 ± 0.45 mm increase in KGW, respectively. Meanwhile, the graft shrinkage in test and control groups was 23% and 21% respectively and there was no statistically significant difference between the groups (P > 0.05).

There was no statistically significant difference between the two groups with respect to the amount of inflammation, foreign body reaction, and keratinization (P > 0.05).

Histologic evaluation revealed 8 samples with Grade 0, and 2 samples with Grade 1 inflammation in the FGG group, whereas in the XADM group, 7 samples with Grade 0 and 3 samples with Grade 1 inflammation were detected. The test and control group showed no significant difference when using Wilcoxon test (P > 0.05).

None of the samples in the both groups had Grade 2 inflammation, also no foreign body reactions were seen in histologic sections. Para-keratinized epithelium was seen in all specimens Figure 4. Figure 4

(a and b) Stratified para-keratinized epithelium at 6 month (arrow head), blood vessels and normal connective tissue with minimal inflammatory infiltrate and collagen fibers which oriented normally observed in all specimens of the xeno (ostrich) acellular dermal matrix and free gingival graft groups. The presence of apparent capillaries and randomly oriented collagen bundles are as signs of the connective tissue regeneration by xeno (ostrich) acellular dermal matrix as a template or scaffold; (a) = Xeno (ostrich) acellular dermal matrix(hematoxylin and eosin stain; original magnification: ×160); (b) = Free gingival graft(hematoxylin and eosin stain; original magnification: ×160)

FGG procedure due to its limitations made clinicians' attention toward use of substitute materials. ADM has been used as dermal substitute however, there were also limitations. ⁹ Thus, ADM from xenogenic skin was introduced. Porcine ADM, as a temporary treatment has been used clinically in burns, mucogingival defects, ⁶ , ¹⁵ and other skin wounds. ⁹ , ¹⁶

Recently, xenogenic skin of ostrich has offered fruitful results in the treatment of deep dermal burns. ¹³ The results of histological studies on structure of ostrich skin showed that this material in both dermis and epidermis level, is very different from those found in other domesticated animals. ¹⁷ , ¹⁸ The dermis is a very dense connective tissue predominantly composed of collagen. ¹⁷ Since, collagen is a key component in healing wounds, ⁵ the development of dermal connective tissue makes ostrich skin highly suited for studying wound healing and skin grafting. ¹³ , ¹⁶ , ¹⁷ , ¹⁸ In ostrich skin, the stratum compactum is a dense layer of connective tissue that predominantly consists of collagen. ¹⁶ Moreover, there is a thin and highly vascularized layer of loose connective tissue between the stratum superficiale and stratum compactum; despite our little knowledge; authors showed that XADM has a dense but porous structure that predominantly composed of collagen. It works like a biologically compatible framework that let fibroblasts, epithelial cells and blood vessels migrate and easily adhere to it, through which a newly formed tissue appears. ¹⁶ , ¹⁷

While the results of our study showed significant increase in the KGW, there was no statistically significant difference between the study groups; thus, treatment by ostrich skin could result in increase of KGW similar to FGG.

The graft shrinkage rate in the ADM site was a little greater than in the FGG site (23 and 21 percent respectively) however, there was no statistically significant difference between the two groups. It is similar to the results of Harris which showed the amount of increased keratinized tissue at the ADM sites was similar to that in FGG-treated sites (4.1 mm), albeit the mean dimension of the grafts was not reported. ⁵ Wei et al. compared the ADM and FGG and reported significant increase in width of keratinized tissue with FGG rather than with ADM, however, the graft shrinkage in ADM group was greater than FGG group (71% vs. 16%). ¹⁹ This difference between the studies might be attributed to graft location, observation period, surgical technique, and the graft extent. It was showed that the thickness of the grafts are important, the thick grafts show statistically the least shrinkage. ²⁰ , ²¹ , ²²

Thoma et al., in their systematic review article on soft tissue grafting techniques revealed statistically significant less shrinkage in FGG group. ²³ More shrinkage of ADM allograft in comparison to autogenous tissue may be due to the ADM fabrication process, since this material was prepared from cadaver skin after removal of epidermis and other cellular components. ¹⁵ , ²¹ , ²³

In our study, the mean shrinkage values in different intervals were respectively lower than those in studies done by using allogenic ADM. ¹⁹ , ²⁴

In the absence of dermal matrix, fibroblasts initially synthesize an immature matrix which subsequently are remodeled to form a hypertrophic scar or scar contracture. ²¹ , ²³ The researchers suggested that after removing the cellular components (decellularization), structural and functional molecules such as collagen and sulfated glycosaminoglycans (sGAG) remain, this improve and facilitate the communication of the adjacent cells and their external environment. It was shown that inducing regeneration and delay in wound contraction were impacted by sGAG. ²⁴ , ²⁵ , ²⁶

Independent photographic analysis of the treated areas in our study showed XADM as a good model in increasing keratinized gingiva rather than FGG. It is also preferred in terms of tissue color match with adjacent untreated tissue and absence of scar tissue or keloid-like appearance. There was an increase of collagen in XADM sites, but the shape and distribution of collagen were similar to FGG sites, however, it was neither similar to healing process with ADM, nor with the hyalinized collagen seen in keloid scars Figure 4. Hence, our findings are similar to the results of Scheyer and contradict Wei studies. ⁴ , ¹⁹

Moreover, mild inflammatory cell infiltration was observed in XADM and FGG-treated sites 6 months after treatment in histologic specimens of our study. The presence of inflammatory cells is important in continuity process of ADM with host tissues, that is, generation of a tissue more similar to the host tissue. ²⁷

After 6 months, blood vessel penetration could be seen in XADM, also collagen fiber bundle branches from XADM to the connective tissue were seen in all directions, XADM appeared well integrated with the host connective tissue. Histological presence of mild inflammatory cells and new vessels formation beneath ostrich acellular matrix in our study agreed with Farahany results. ¹³

To the best of our knowledge, it is the first study reporting the efficacy of the ostrich acellular dermal matrix in increasing the band of keratinized tissue in a dog model study. In this animal study, a good healing pattern and clinical behavior were seen in the matrix along with similar clinical outcomes when compared with the standard FGG. It also demonstrated acceptable increase of keratinized tissue, and good maintenance of the marginal tissue health with better color match.

Interpretation of these results was limited by certain aspects of the study design. Although the duration of this study was typically the same as for soft tissue studies, ²³ the longer follow-up study would have allowed us to more precisely evaluate the durability of the result. Moreover, there were several limitations of the esthetic assessment such as the nature of the photography (as opposed to direct assessment), as well as varying degrees of consistency in the photographs (e.g. lighting, focus, global vs. close-up views).

The results showed similar outcomes after treatment with autogenous FGG and XADM in terms of increasing the width of keratinized tissue, the amount of shrinkage, and the quality of the attached tissue after 6 months' follow-up. Due to FGG disadvantages, XADM seems to be a suitable alternative for FGG. Since, there is no similar study on XADM with ostrich origin in periodontics, it is suggested:

More histologic emphasis on matrix characteristics including collagenous and noncollagenous proteins (e.g. tenascin, elastin,)

Financial support and sponsorship

Nil.

Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non-financial in this article.