To what extent does hyaluronic acid affect healing of xenografts? A histomorphometric study in a rabbit model

ABSTRACT Among the many graft materials that have been used for the treatment of bone defects in oral and maxillofacial regions is xenograft. To improve osteoconductive effects of xenografts, they have been combined with various biocompatible materials, such as hyaluronic acid and bone morphogenetic protein. Objective To determine bone-healing capacity of high molecular weight hyaluronic acid (HA) combined with xenograft in rabbit calvarial bone defects. Material and methods Ten adult male New Zealand rabbits (mean weight 3 kg) were included in the study. Three 6-mm-diameter bicortical cranial defects were created on calvarial bone of all rabbits. These defects were filled as follows: a) xenograft; b) HA+xenograft; c) autograft. One month after the first operation, rabbits were sacrificed. Specimens were evaluated histomorphometrically. Results Considering multiple comparisons, differences regarding new bone were statistically significant between all groups (p<0.05). The volume of residual graft was significantly decreased in HA group compared to xenograft group (p=0.035). Marrow space, trabecular thickness (TbTh), trabecular width (TbWi), trabecular separation (TbSp), and number of node: number of terminus (NNd:NTm) in the autograft group were significantly better than xenograft and HA groups (p<0.05). However, regarding marrow space, TbTh, TbWi, TbSp, and NNd:NTm values, xenograft and HA groups showed similar results and the difference were not significant (p>0.05). Conclusion These results support that high molecular weight hyaluronic acid could contribute to the healing of xenograft by improving the percentage of new bone formation and reducing the percentage of residual graft. However, HA did not significantly affect the quality of newly formed bone assessed by microarchitectural parameters.


Introduction
An understanding of the mechanisms of bone repair and regeneration is basic to bone defect treatment, alveolar socket healing and dental implant surgery 2 .
Thus, various graft materials such as autografts, allografts, xenografts, and alloplasts have been  which was, subsequently, shaved and stained with iodine solution to avoid any bacterial contamination.
We exposed the calvarial bone (occipital, frontal, and parietal bone) by making a full-thickness linear incision from nasal bone to mid-sagittal crest. Afterwards, we created three 6-mm-diameter bicortical cranial defects by using trephine bur under copious irrigation with sterile saline solution. To prevent damage to the durameter, first we created a portion of cavity depth with the trephine bur and afterwards we removed the remaining portion of the cavity with the diamond round bur. In conclusion, three bicortical defects were obtained. These defects were filled as follows: The ratio between the numbers of nodes and termini in a section is an index of the spatial connectivity in the trabecular network. Except for NNd:NTm, which was expressed in ratio, all parameters were expressed in µm.

Results
After surgery, all rabbits recovered well and the expected increase in weight during the post-surgical period was recorded. Complications, such as paralysis, convulsions, respiratory distress, and wound infections were not observed. Results of histomorphometric comparisons among groups are shown in Table 1.
Regarding percentages of tissue compartments and microarchitectural parameters, significant differences were observed among study groups (p<0.05).  regarding new bone between all groups were also statistically significant (p<0.05). New bone value was best in the autograft group (p<0.05). New bone value of the xenograft+HA group was also significantly greater than the xenograft group (p<0.05). The volume of residual graft was significantly decreased in HA group compared to xenograft group (p=0.0355) (Figures 2, 3, and 4). Therefore, we created the three 6-mm-diameter bicortical defects on the calvariae of the rabbits to test whether HA accelerates the healing of xenogenic graft material. These defects were filled with the following materials: xenograft; a combination of xenograft and high molecular weight HA; and autogenous graft. Our rationale for using high molecular weight HA in this study is that Sasaki and Watanabe 24 (1995) reported that high molecular weight HA increased new bone formation in rat femoral defects after bone marrow ablation.

Considering multiple comparisons, differences
The treatment of craniomaxillofacial defects is one of the greatest challenges in bone healing. Autografts induce several bone-healing mechanisms, such as osteogenesis, osteoinduction, and osteoconduction 6,8 .
Although xenografts are widely used in dentistry, they have a limited capacity for osteogenesis because of their low viable cell capacity. In addition, the chemical processes required to inhibit the transmission of animal diseases prevent xenografts from having osteoinductive effects 13,21 . Xenografts that have no osteogenic or osteoinductive effects lead to poor clinical results. However, modern tissue engineering technology makes it is possible to design new scaffolds and tissue grafts to improve osteogenic, osteoinductive, and osteoconductive effects and to mitigate certain disadvantages of these grafts 21 .
Owing to its regulatory role in bone and fracture healing, HA is used to increase scaffold production in tissue engineering 21 . In our study, the application of xenograft mixed with HA into the defect was easier Scaffolding materials such as HA are also known to have osteogenic and angiogenic potential because of their high vascular and cellular activity 11 .
In addition to new bone formation, which is an important indicator in the complete regeneration of bone defects, the microstructure and microarchitecture of newly formed bone are significant indicators of bone quality 10,17 . In this animal study, three different treatment modalities were compared, and obtained specimens were analysed regarding marrow space, trabecular thickness, trabecular width, trabecular separation, and node:terminus ratio, which are essential histomorphometric parameters of bone quality. A node in the network of bone tissue is a connection point of three or more trabeculae; however, a terminus is described as a point at which a trabecula is not joined to any other one. It has been supposed that the NNd:NTm is a way of explaining the connectivity of the trabecular network 30 .
In this study, the structures of bone microarchitecture and the marrow space of the autograft group were significantly better than in the other two groups.

Conclusions
These results support our hypothesis that the high molecular weight HA can positively affect anorganic bovine bone healing concerning new bone formation in a short time; however, HA does not appear to significantly affect the structure of the trabeculae.
There is a need for further studies to evaluate the microarchitecture of newly formed bone tissue during xenograft healing when combined with the high molecular weight HA.

Conflict of interest
Authors have no conflict of interest regarding any financial and personal relationships with other people or organisations that could inappropriately influence this study.