Immunohistochemical evaluation for integrin binding sialoprotein on healing process of intrabony defect treated by bone sialoprotein

: Background: Bone defect healing is a multidimensional procedure with an overlapping timeline that involves the regeneration of bone tissue. Due to bone's ability to regenerate, the vast majority of bone abnormalities can be restored intuitively under the right physiological conditions. The goal of this study is to examine the immunohistochemistry of bone sialoprotein in order to determine the effect of local application of bone sialoprotein on the healing of a rat tibia generated bone defect. Materials and Methods: In this experiment, 48 albino male rats weighing 300-400 grams and aged 6-8 months will be employed under controlled temperature, drinking, and food consumption settings. The animals will be subjected to a surgical procedure on the medial side of the tibiae bone, with the bone defect repaired with absorbable hemostatic material in the control group (12 rats). The experimental group (12 rats) will be treated with local administration of 30 μl bone sialoprotein fixed by absorbable hemostatic sponge. After surgery, the rats will be slaughtered at 7, 14, and 28 days (four rats for each period). Results: Immunohistochemical analysis of bone sialoprotein by stromal cells reveal a substantial difference between the bone sialoprotein group and the control group. Conclusion: The study concludes that local application of bone sialoprotein could be a successful therapeutic treatment for bone injuries; these findings are encouraging for future clinical use.


Introduction
The organic matrix of the bone, of which type-I collagen is the main component, which is largely responsible for bone repair. The involvement of non-collagenous proteins (NCPs) in bone deformation, on the other hand, is not well understood. The majority of NCPs are found in far lower concentrations than collagen and are known to influence bone mineralization, participate in cell signaling, and they have hormonal roles (1,2) . NCPs such as bone sialoprotein (BSP) , osteopontin ( OPN)and osteocalcin (OC)have recently been shown to improve bone fracture resistance. Diffuse damage arises in rat and human bone, allowing bone to disperse energy without producing overt fracture (3) .
Non-collagenous extracellular bone-matrix proteins like bone sialoprotein are synthesized and secreted by osteoblasts. These non-collagenous matrix proteins are recognized to play a key role in the mineralization of the bone (4,5) . These proteins found in the bone matrix have proven to be particularly valuable as osteogenic indicators (6,7) .
Bone sialoprotein is a non-collagenous extracellular matrix protein that is highly expressed by osteoclasts, osteoblasts and hypertrophic chondrocytes in bone, and is especially abundant in primary bone formation locations (8,9) .
Bone sialoprotein is a powerful mineralization nucleator as well as a matrix-associated signal that promotes osteoblast development and enhanced mineralized matrix synthesis (10,11) It is abundantly expressed in bone by osteoblasts, osteocytes, osteoclasts, and chondrocytes (12,10) . BSP overexpression boosts osteoblast-related gene expression and accelerates mineralized nodule formation in culture, despite the fact that it has long been thought to be a hallmark of late osteoblastic differentiation. In contrast, inhibiting the expression of osteoblast markers by reducing BSP expression in osteoblasts with particular shRNA leads to a considerable reduction in matrix mineralization (10) .

Materials and methods
All experimental procedures are carried out in conformity with the Baghdad College of Dentistry's ethical principles. In this investigation, 48 albino male rats weighing 300-400 grams and aged 6-8 months will be employed under strict temperature, drinking, and food consumption controls. The animals are given an intrabony defect in the medial side of the tibiae bone, which is treated with absorbable hemostatic sponge in the control group (12 rats) and local application of 30 μl bone sialoprotein fixed by absorbable hemostatic sponge in the experimental group (12 rats). The rats were slaughtered seven, fourteen, and twenty-eight days following operation (four rats for each period in each group).

Surgical technique
A surgical procedure was performed on the animals. The procedure was done in a sterilized environment with a gentle technique. The dose of general anesthesia given to each animal was calculated based on its weight. Intramuscular injections of xylazine 2% (0.4 mg/kg B.W.) and ketamine HCL 50 mg (40 mg/kg B.W.) were used to induce general anesthesia, as well as an antibiotic treatment with oxytetracycline 20% (0.7ml/kg) intramuscular injection. Tibiae were shaved, and the skin was washed with a solution of ethanol and iodine, followed by an alcohol-soaked piece of cotton. An incision was created, and the skin was removed. The skin and fascia flap were mirrored after an incision was made. A hole of 1.8mm was drilled with a tiny spherical bur at a rotational speed of 1500 rpm using instrument drilling and continuous cooling with irrigated saline.
The operation site was cleaned with saline solution after the hole preparation to eliminate debris from the drilling site. After the operation, the area was air dried, and then 30 μl bone sialoprotein was applied to the experimental group. The muscles were sutured using absorbable cat gut, then the skin was sutured. A local antibiotic was applied on the surgical site (tetracycline spray).

Results
The presence of a brown cytoplasmic stain indicated a positive reading, whereas the absence of immunological reactions suggested a negative reading, depending on the positive and negative controls.

Analytical statistics
Mean, S.D., Min., Max., F-test, P-value were used to assess bone cell count as well as osteoblast, osteocyte, and stromal cells expressed by bone sialoprotein.

Results of immunohistochemistry
In the control group, bone sialoprotein expression was as follows: At 7 days, immunohistochemical analysis of the control group revealed positive expression in osteoid tissue, osteoblast, and osteocyte. Basal bone expression is negative (Figure 1). Bone sialoprotein expression is positive in osteoblast, osteocyte, and bone marrow stromal cells after 14 days (Figure. 2). Positive expression was detected in osteoblasts, osteocytes, and osteoclasts after 28 days. The new bone has a negative BSP expression (Figure 3).

Bone sialoprotein expression in the bone sialoprotein-treated group:
Positive staining in the osteoid tissue is after 7 days. Basal bone expression is negative (Figure 4). A positive DAB stain for BSP was seen in stromal, osteoblast, and osteocyte cells after 14 days ( Figure 5). BSP immunohistochemistry expression in osteoblasts and osteocytes after 28 days. BSP expression was negative in mature bone, which contain several tiny Haversian canals ( Figure. 6).
Statistical analysis revealed a high significant value for bone sialoprotein group in comparison to control and in periods (7,14, and 28 days) as indicated in Table (1). for positive expression of bone sialoprotein by stromal cells in study groups.
Positive expression of bone sialoprotein by osteoblasts, osteocytes, and osteoclasts, as determined by the following statistical analysis are as follow: (See Table (2).
-In 7 days, osteoblast and osteocyte levels were highly significant, while osteoclast levels were nonsignificant.

Discussion
The goal of this study was to see how local application of bone sialoprotein affected experimentally produced bone defects in rats' tibiae. Over the last decade, the rat has been employed in a considerable fraction of animal fracture investigations in major orthopedics research (13) .

B B
Proliferation, apoptosis, adhesion, migration, angiogenesis, and ECM remodeling could all be influenced by bone sialoprotein (14) . In a nude rat model, anti-BSP Ab was also found to prevent osteolysis while stimulating bone growth (15,16) .
Bone sialoprotein expression and localization were brownish in color in all groups, showing that there was expression of bone sialoprotein; positive cells were found at osteoblast, osteocyte, and osteoclast in all groups. The new bone matrix and the surrounding area of the resorptive lacunae showed high positive expression for bone sialoprotein. This was in line with the findings of (17,18) , and (19) , who claimed that strong BSP staining in newly produced bone in swine fetuses' mandibular alveolar bone. In this investigation, bone sialoprotein was expressed in a large number of osteoblasts, osteocytes, and a small number of osteoclasts in the space of a bone defect for 1 week in all groups. This conclusion matched with (20) , who showed significant staining of osteoblasts and osteocytes associated with newly produced bone during periodontal regeneration in dogs. While disagreeing with (21) who claimed that faint BSP staining in osteoblasts present in embryonic rats' mandibles.
In the experimental group, however, there was a decrease in bone sialoprotein expression over time. This finding is consistent with (22) who found that newly formed bone had more intense BSP staining at 1 week, whereas mature bone was weakly stained, and osteoblasts and osteocytes had higher BSP positivity at 1 week, which gradually decreased with time when staining was mild and limited to the lacunae. Also (23) Found that the bone sialoprotein mRNA signal was strongest in osteoblasts along the surface of woven bone trabeculae in actively growing bone tissue, and that it decreased with bone growth.
These findings suggest that while bone sialoprotein is necessary for de novo creation, it is less important for bone growth because growth is still occurring.

Conclusion
The findings of this investigation reveal that bone sialoprotein has the potential to aid in the mending of bone abnormalities.