Osteoinduction Using a Prefabricated Scaffold in the Gluteus Muscle of Wistar Rats: An Animal Study

Abstract

Introduction:

Bone grafts are very significant for the reconstruction of the trauma and the deformations created thereafter. Various bone forming and promoting agents are widely studied. Hence, in this study, we aim to evaluate the osteoinduction effect of scaffold impregnated with Simvastatin in wistor rats in an observational study.

Material and Methods:

We implanted 18 Wistar rats to be equally divided as controls and test groups. Polycaprolactone coated with 20 mg concentration of Simavastatin under sterile condition was used as test and hydroxy appetite as control. Then, we observed the histopathological Heamatoxilin and Eosin and ABH, sections at 4, 12, and 26 weeks. We noted the calcium deposition and bone formation.

Results:

We observed a significant variation among groups at 4, 12, and 26 weeks in the calcium deposition. While for the bone formation no significant variation were noted among groups at 4 and 12 weeks, however, there was a significant difference noted at 26 weeks. At the end of 12 weeks, mild fibroblast proliferation was seen in the surrounding area. After 26 weeks, ABH sections showed focal area of fibroblast proliferation with mild mononuclear infiltration was noticed; but implant could not be located.

Conclusion:

We conclude that Simavastatin showed an anabolic effect on bone healing.

INTRODUCTION

The increase in the automobile and the transport facilities have inadvertently increased the accidents. The trauma may lead to bone fractures and also considerable disfigurement. Graft placement is one of the unpredictable tasks the surgeons find. Various specialities of the surgery use the bone grafts, especially the plastic surgery and maxillofacial surgery.[1] There are various types of the bone grafts ranging from autogenous to newer graft materials. However, the autogenous grafts are still the preferred grafts for the obvious reason of acceptability. The auto grafts though are first option may require an additional surgery for the procurement of the graft. Also they may lead to the considerable scar at the site of the graft removal.[2] There are various methods in the placement of the grafts. These various grafts may act in an osteoconductive or the osteoinductive manner. Recently, various pharmaceutical agents are used in the grafts for their osteoinductive effects. In the study conducted by Mundy et al.,[3] they found that statins bring about the osteoinductive effect in the experimental study on rats. The drug that has been classified under statins, that is a modification of lovastatin, Simvastatin has shown to be beneficial in the bone growth. This drug has been shown to increase the mineralization by increasing the alkaline phosphatase activity. Simvastatin lowers the levels of the interleukin 89, thus lowering the inflammation. Simvastatin enhances the type I collagen, bone osteocalcin, and sialoprotein. This drug is poorly available to the bone morphogenic cells if taken orally.[4,5] Hence, in this study, we aim to evaluate the osteoinduction effect of scaffold impregnated with Simavastatin in wistor rats in an observational study.

MATERIAL AND METHODS

In this study, we selected 18 Wistar rats of either sex weighing approximately 180 g and 200 g from the biomedical technology division, animal sciences lab, Sree Chithra Thirunal Institute for Medical Sciences and Technology, Kerala, India. The institutional ethics committee clearance was obtained for our study. Only water and the food pellets were given to the rats. The cages were numbered and appropriately labeled. Animals were kept in a monitored environment air conditioned to a temperature of 22 ± 3° C, humidity of 30% to 70%, and a light dark cycle of 12 h, and both the male and the female rats were employed in the study.

Making of material for the tests

Polycaprolactone (PCL) coated with 20 mg concentration of Simavastatin under sterile condition was used as test and hydroxy appetite as control. PCL is commercially available as a large mesh that was cut down to 1 × 10 mm linear scaffolds using surgical blade.

Surgical methods

We selected healthy adult Wistar rats whose gluteal muscles are sufficiently large in size and the fur was clipped on either side over the gluteal muscle on both sides. The intramuscular implantation was carried out under clean and aseptic conditions. Rats were anesthetized using 2% ketamine and xylazine. The skin of the anesthetized rats was lightly swabbed with 70% alcohol and air dried. An incision was made in the skin over the gluteus muscle. The skin and the subcutaneous tissue were reflected and an incision was made on the muscle. The scaffolds were placed inside the muscle space using surgical forceps. The surgical site was then closed with sterile sutures. The sutures also gave mechanical retention to the scaffold placed. The rats in each group were euthanized later. The test and control implant materials along with the surrounding tissues were explanted. The explanted materials were fixed in 4% buffered formalin (pH 7.4), dehydrated in a graded series of alcohol, and subsequently embedded in paraffin. The paraffin sections were stained with hematoxylin eosin and ABH and were later studied under scanning electron microscope. The implant sites were examined for hemorrhage, necrosis, discoloration, and infection. The control and the test implanted materials along with surrounding tissue were sent for processing.

RESULTS

In the histopathological changes, the following observations were made:

At 4^th week in the control group, implant could be identified, while in the tests, the remnants of implant were observed surrounded.

At the 12^th week in the control, focal mononuclear infiltration is seen with angiogenesis and fibroblast proliferation, while in the tests, certain region show mild to moderate infiltration of mononuclear cells along with fibroblast proliferation.

At 26^th week in the controls, encapsulated fibroblast proliferation and capsules are also seen, while in the tests, mild mononuclear infiltration with angiogenesis is also observed.

In the ABH changes the following observations were made:

At 4^th week in the control group, no indication of mineralization could be appreciated, while in the tests, implant remnant was seen with adjacent muscle showing coagulation necrosis.

At the 12^th week in the control, mild fibroblast proliferation is seen, while in the tests, blue gray color cellular refractile substance is seen (mineralized areas).

At 26^th week in the controls, focal area of fibroblast proliferation with mild mononuclear infiltration is noticed, but implant could not be located, while in the tests, implant could be identified as a localized area containing multiple cysts; there is fibroblast proliferation around blue to gray stained refractile material, and mild to moderate infiltration of mononuclear infiltration is also noticed.

We observed a significant variation among groups at 4, 12, and 26 weeks in the calcium deposition [Table 1 and Graph 1].

Table 1.

Comparison of the significance of the calcium deposition among groups

Time duration	P
At 4 weeks	=0.002
At 12 weeks	=0.002
At 26 weeks	=0.002

Graph 1.

Comparison of the calcium deposition at 4, 12, and 26 weeks

While for the bone formation, no significant was observed. We observed a significant variation among groups at 4 and 12 weeks; however, there was a significant difference noted at 26 weeks [Table 2 and Graph 2].

Table 2.

Comparison of the significance of the bone formation among groups

Time duration	P
At 4 weeks	=0.455
At 12 weeks	=0.061
At 26 weeks	=0.015

Graph 2.

Comparison of the bone formation at 4, 12, and 26 weeks

DISCUSSION

Many bone morphogenic materials are being studied by the scientists to identify their true potential to be used in the surgical branches. There are some drugs that have shown effect on the bone formation or the acceptance of the graft if given orally.[6] However, there are few that have significantly enhanced the bone deposition when placed in the vicinity of the graft area. In this study, we aim to evaluate the osteoinduction effect of scaffold impregnated with Simavastatin in Wistor rats in an observational study. The osteogenic potential of various biomaterials has been first described in 1883. There are several studies that were done to assess the bone depositions that were seen in the ectopic implantations of the materials. These were either placed in the scaffolds or directly placed in the site of the interest.[7,8,9,10] Some authors have included the “Growth factors” also along with the biomaterials. The osteogenic potential has been first described in 1883. The chief drawback with the conventional grafts like the autograft and the allograft, that is, an extra preparation for the surgery, is the second defect at the donor site, which may be overcome with the newer materials. At least for now, these materials are used in conjunction with the conventional grafts for better acceptance and the quick healing. These processes may be by either the osteoinductive or the osteoconductive effects.[4,8] In our study, we observed that Simavastatin showed an anabolic effect on the bone healing. We can present that our study is one of the firsts to use Simavastatin-impregnated PCL scaffolds to assess the osteoinductive property of combination scaffold. The material we used, PCL, may be considered as a resorbable implant biomaterial that can be utilized as a mesh in craniofacial surgeries due to its osteoconductive ability, as an adhesion barrier, suture material, drug delivery device, etc.[2,3] It is easily degraded, biocompatible, and can be easily handled. The implants in our study were sutured rather than placed in the pockets. The drug-impregnated PCL can migrate into a different site owing to its flexible nature and the agility of the animal. While the non-resorbable sutures served this cause, in our case, it might have also led to inflammatory reactions and hence the presence of inflammatory cells in Heamatoxilin and Eosin (HPE) sections. Much precautious measures would have made our results more obvious. The combination scaffold of PCL with Simvastatin dose might have also lead to inflammatory cells in HPE. At the end of 12^th week, ABH sections showed very obvious lamellar pattern of bone formation on four of the six slides. However, all the sections showed calcium deposition in consistent with calcium deposition of 4^th week. Saraf et al.[9] in their study found bone remodeling and repair in 4 weeks and increased stiffness and strength in 8 weeks. The hydroxy apetite-controlled sections of 12^th week showed moderate inflammatory cells in histopathologic sections without any sign of bone formation. This leads us to the point that Simavastatin has got significant osteoinductive property. On the 26^th week, five of the sections showed lamellar pattern of bone formation. But the presence of osteoblasts cells could not be substantiated. The hydroxyl apetite control sections of 26^th week showed severe inflammatory cells. Taking into account the consistent mineral deposition on the test sections and the lamellar pattern of bone formation that charted from the 4^th week and were prominent at the end of 26^th week, its substantiated that Simavastatin granule-impregnated PCL scaffold holds a significant potential for osteoinduction. In test slides on 4^th week ABH, all the six test sections gray colored material was noted indicative of mineralization that was recognized as calcium deposits. Two of the sections among them showed significant lamellar pattern of bone formation. However, the presence of osteoblast cells was not clearly identifiable. This is in contradiction to the study by WU et al. in which they found Simavastatin-promoted bone formation via increase in osteoblasts numbers and matrix protein levels at the end of 6 weeks. The hydroxyl apetite control sections of 4^th week showed mild inflammatory cells. The flaws in measuring the concentration of Simavastatin just before implantation, the inflammation caused by non-resorbable sutures, and the use of two different materials as test and control can be suggested as our setbacks during this course of this experiment. The study can be continued on a larger animal for a longer period of time in the upcoming days to know whether the use of quantitatively and qualitatively analyzed Simavastatin granules impregnated on PCL of different shapes on a larger animal can generate clinically persistent bone.

CONCLUSION

Simvastatin aided in the repair of the defect. It also lowered the inflammation by its osteoblastic activity. It also aided in rats, in the matrix release from its ability to produce the lamellar bone structures. We conclude from our study that Simavastatin showed an anabolic effect on the bone healing.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.