|Year : 2020 | Volume
| Issue : 1 | Page : 25-30
Evaluation of the effect of β-tricalcium phosphate nanoparticles on bone defect healing in diabetic rats
Wael S. Ahmed, Hany A. Sherif, Galal A. Nasr PhD, Lecturer P.H.D
Department of Oral Biology, Faculty of Dental Medicine, Al-Azhar University (Males), Cairo, Egypt
|Date of Submission||27-Aug-2019|
|Date of Acceptance||30-Jan-2020|
|Date of Web Publication||20-Jun-2020|
Galal A. Nasr
Department of Oral Biology, Faculty of Dental Medicine, Al-Azhar University (Males), Cairo, Kafr Elzayat, Elghrbia 31611
Source of Support: None, Conflict of Interest: None
The aim of this study was to evaluate histologically the bone defect's regeneration after application of β-tricalcium phosphate nanoparticles (Nβ-TCP) intibial bony defects of diabetic rats.
Materials and methods
Sixteen adult male albino rats were used and their weight was almost 250 g. The animals were grouped into two equal groups: group 1 (control diabetic) which had not receive the graft; group 2 (experimental diabetic) which had received the graft material (Nβ-TCP). Specimens were harvested on days 7 and 28 after surgical procedures, prepared, stained with hematoxylin and eosin, and examined histologically.
The histological examination demonstrated obvious retardation in granulation tissue formation, organization and bone formation in the control diabetic group 1 than group 2 along the different intervals of this study. This retardation in the healing of diabetic contro1group was due to the effect of diabetes as it reduced cellular proliferation in early callus, collagen synthesis content, osteoblastic activity and reduced bone mineralization. Moreover, there was great acceleration in granulation tissue formation, organization and bone formation in experimental diabetic group 2 which received the graft material.
Nβ-TCP has been shown to have a good biocompatibility and osteoconductivity and can be used to enhance bone healing specially in retarded conditions as in diabetes mellitus.
Keywords: β, calcium phosphate nano, diabetic, tri
|How to cite this article:|
Ahmed WS, Sherif HA, Nasr GA. Evaluation of the effect of β-tricalcium phosphate nanoparticles on bone defect healing in diabetic rats. Tanta Dent J 2020;17:25-30
|How to cite this URL:|
Ahmed WS, Sherif HA, Nasr GA. Evaluation of the effect of β-tricalcium phosphate nanoparticles on bone defect healing in diabetic rats. Tanta Dent J [serial online] 2020 [cited 2020 Sep 25];17:25-30. Available from: http://www.tmj.eg.net/text.asp?2020/17/1/25/287097
| Introduction|| |
Diabetes mellitus (DM) defined as a group of metabolic disorders characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. It is caused by either autoimmune destruction of insulin-producing cells (type I) or resistance of the body to insulin (type II) . Poorly controlled diabetes is associated with adverse systemic sequelae including increased susceptibility to infection, delayed wound healing and microvascular complications that lead to decreased immune response . Hyperglycemia produces deleterious effects on bone matrix and its components, and also affects adherence, growth and accumulation of extracellular matrix . Many mechanisms have been reported by which diabetes may affect bone including inhibition of both osteoblast differentiation and expression of growth factors that promote bone formation and an increased osteoclast proliferation, resulting in lower bone mineral density and increased risk of fractures .
Various calcium phosphate biomaterials had been developed for use as bone substitutes owing to their biocompatibility and osteoconductivity, one such material, β-tricalcium phosphate (β-TCP), had been shown to have good biocompatibility and osteoconductivity in both animal experiments and clinical settings . A report has described osteogenesis by calcium phosphate alone after implantation in soft tissues , and such osteoinductivity is a desirable characteristic of a bone-substitute material.
The nanotechnology is fast approaching, which was unheard of two decades ago. The growing interest in the nanotechnology field has given emergence to the new field of 'nanomedicine,' the science and technology of diagnosing, treating, preventing diseases, preserving, and improving human health . Nanotechnology has allowed for the possibility of fabricating nanophase particles with particle size less than 100 nm and functions suitable for targeting and treating various bone diseases . These nanomaterials have shown superior properties compared to their conventional (or micron structured) counterparts due to their distinctive nanoscale features and novel physical properties ,. Nβ-TCP granules with higher phase purity (>99%), better mechanical compressive strength (>2.22 MPa), higher porosity (>75%) including macropores, mesopores, and micropores, and larger specific surface area (>2.50 m 2/g) were successfully fabricated from wet chemically precipitated method . Conventionally, the nano-sized particles in porous granules could play a significant role in increasing porosity and specific surface area of granules. Furthermore the presence of macroporous (>100 μm) and mesoporous structure (10–100 μm) favors cell ingrowth and newly bone formation while the microporosity (<10 μm) allows the penetration of the body fluids into implant .
Typically, it had been reported that both the mesopores (10–100 μm) and the macropores (>100 μm) play a significant part in stabilization of the initial blood clot and subsequent vascularization and integration of the material in the bony tissue . In particular, the vascularization is important for successful bone regeneration, especially when using a biomaterial because of its role in the nutrition of the migrating cells . The microporosity also can be a strategy to manipulate resorption and dissolution rate; greater microporosity means greater degradation rate ,. In particular, it has also been demonstrated that the large specific surface area which can be achieved by increasing the number of micropores is essential for the osteoconductivity for bone regeneration ,,,,,.
The purpose of this study was to evaluate histologically the effect of nano-sized particles of β-tricalcium phosphate on healing of tabial bony defects of diabetic rats.
| Materials and Methods|| |
This study was carried out on 16 adult male albino rats; their average weight was about 250 g. The procedure management was carried out at house animals of Faculty of Medicine, Cairo University, Egypt according to the principles of ethical committee of Faculty of Medicine, Cairo University.
Induction of diabetes
Diabetes was induced by a single intra-peritoneal injection of 120 mg/kg monohydrate alloxan dissolved in sterile 0.9% saline. Rats had been fast for 12 h, followed by alloxan administration. After 12 h 10% glucose solution was offered to the animals to prevent hypoglycemia. After 7 days, blood samples were collected from the caudal vein of the animals for evaluation of plasma glucose levels. The animals with glycemic level higher than 250 mg/dl, were considered diabetic and used in this study.
The graft material
Nano β-tricalcium phosphate material was synthesized at Biomaterial Department, Faculty of Engineering, Al-Azhar University (Males), Cairo, Egypt.
The animals were weighed, premedicated by atropine (IM 0.04 mg/kg) and anaesthetized intramuscularly with a combination of 2% xylazine in a dose of 5 mg/kg (ADWIA, 10th of Ramadan city, Egypt) and ketamine in a dose of 50 mg/kg (ROTEXMEDICA, Bunsenstrasse 4 Trittau, Germany). The overlying skin of the tibia was shaved and disinfected with iodated alcohol. An incision about 1.5 cm was then made in the medial aspect of the tibia, full thickness flap, the skin, subcutaneous tissue and muscular layer were reflected exposing the tibial bone. A circular 4-mm diameter defect created using a sterile carbide rose head surgical bur mounted in a dental hand piece connected to a micromotor with 2000 rpm speed, a metal template with a round cavity 4 mm in diameter had been used to standardize the defect site and size. The process of defect creation was done under copious irrigation with warm saline to avoid bone burning and to maintain the vitality of bone cells around the defect.
In group 1 the defect will be left empty without graft, while in group 2 the graft material was placed in the defect, then the flap repositioned, the muscular layer was sutured with resorbable #4.0 catgut and the skin was sutured with interrupted #4.0 silk sutures.
The animals were divided randomly into two equal groups as follows:
- Group 1: control diabetic group which had not received the graft.
- Group 2: experimental diabetic group which had received the graft material.
Two animals from each group were sacrified after 7 and 28 days postoperatively. Bone regeneration at the defect area was assessed histologically using hematoxylin and eosin stain at ×400.
| Results|| |
Seven days interval of group 1 demonstrated blood clot filling the defect which infiltrated with inflammatory cells mainly macrophages engulfing the clot. No signs of granulation tissue formation was present and no bone resorption at the side of the defect [Figure 1] and [Figure 2].
|Figure 1: Histological section of group 1 at 7 days interval showing: old cortical bone (OB), blood clot (BC), inflammatory cells infiltration (arrow head) (hematoxylin and eosin, ×100).|
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|Figure 2: Higher magnification of the previous figure showing: inflammatory cells (black arrows), macrophages (arrow heads) (hematoxylin and eosin, ×400).|
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On day 28 of group 1 there was thick band of typically organized granulation tissues at the surface of the defect which characterized by tightly packed collagen bundles with proliferating blood capillaries. The base of the defect was almost closed by thin trabeculae of woven bone connected to the old bone forming a bridge. Moreover, in some areas bone trabeculae were harboring marrow heamopiotic cavities in between them which filled with haemopiotic tissue. In addition, there was recruitment of osteoblasts within the granulation tissue around the woven bone trabeculae, also there were numerous osteoblasts imprisoned within the bone trabeculae with narrow osteocytic spaces [Figure 3] and [Figure 4].
|Figure 3: Histological section of group 1 at 28 days interval showing: typically organized granulation tissue (GT), old bone (OB), thin bridge of new woven bone (NB), osteocytes with narrow osteocytic space (arrow head), marrow cavity with haemopiotic tissue (arrow) (hematoxylin and eosin, ×100).|
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|Figure 4: Higher magnification of the previous figure showing: typically organized granulation tissue with tightly packed collagen bundles, fibroblasts (arrow head), new woven bone (NB), osteocytes with narrow osteocytic space (thin arrows), recruitment of osteoblasts (thick arrow) (hematoxylin and eosin, ×400).|
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Seven days interval of group 2 demonstrated some remnants of the material (Nβ-TCP) surrounded by well-organized granulation tissue at the surface of the defect. The obvious feature was wide irregular spaces containing remnants of the material mixed with the blood surrounded by fibrovascular connective tissue which infiltrated the macropores of the material. Notable proliferating blood vessels were observed in the granulation tissue. The newly formed granulation tissue was seen and infiltrated by macrophages and multinucleated cells. Moreover, there was recruitment of osteoblasts and at the base and sides of the defect with new woven bone formation demarcated from old bone by a reversal line. This newly formed bone was highly cellular, fibrillar and characterized by imprisoning of multiple osteoblasts with wide osteocytic spaces [Figure 5] and [Figure 6].
|Figure 5: Histological section of group 2 at 7 days interval showing: new bone formation (yellow arrow), wide spaces (SP) surrounded by well-organized granulation tissue (arrow), which infiltrated the macropores of the material, blood vessel (arrow head) (hematoxylin and eosin, ×100).|
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|Figure 6: Higher magnification of the previous figure showing: old bone, new bone formation, well organized granulation tissue (black arrow), reversal line between old and new bone (yellow arrow), recruitment of osteoblasts (blue arrow), osteocytes with wide osteocytic spaces (triple arrow), (hematoxylin and eosin, ×400).|
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On day 28 of group 2 demonstrated that the defect was closed by new woven bone formation with multiple reversal lines and small amount of imprisoning osteoblasts. In as much as there were multiple dispersed bony trabeculae of different sizes and shapes filled the defect, harboring in between haemobiotic tissue with active mitotic index. Moreover there were configuration of primary osteons and narrowed blood vessels. A reversal line demarcating the old bone from the newly formed bone at the periphery of the defect was also observed [Figure 7] and [Figure 8].
|Figure 7: Histological section of group 2 at 28 days interval showing: old bone (OB), new woven bone (NB), marrow cavities with haemopiotic tissue (arrow head), primary osteons (arrows), reversal line (yellow arrow) (hematoxylin and eosin, ×100).|
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|Figure 8: Higher magnification of the previous figure showing: active remodeling appeared by multiple reversal lines (arrows), primary osteon (arrow heads) (hematoxylin and eosin, ×400).|
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| Discussion|| |
At seven days interval there were remarkable histological differences between the groups of this study. The control diabetic group showed blood clot with high infiltration of inflammatory cells with no signs of granulation tissue formation suggesting retardation in healing process. These findings were parallel with Retzepi et al. , and Stolzing et al. , who stated that diabetes, reduced cellular proliferation in early callus and reduced collagen synthesis content. Likewise Andriankaja et al. , Pacios et al. , referred the retardation in bone regeneration to the prolonged inflammation associated with DM. They added that diabetics have difficulty in down regulating inflammation once induced. Moreover, increased levels of tumor necrosis factor may limit the capacity of diabetics to downregulate other inflammatory genes and increase apoptosis, which has been shown to reduce bone coupling in diabetic animals.
While at the experimental diabetic group there were thin rim of well-organized granulation tissue surrounding wide irregular spaces containing remnants of the material dissolved with the blood. Furthermore, there was new woven bone formation within the macroporosity created by the material, the new bone was highly cellular and fibrillar due to rapid formation. Our findings were similar to what reported by Robert et al. , who observed several large spaces which represent β-TCP lost during decalcification and there was pink stained material inside these spaces suggesting that material 'fibrin or proteins' had penetrated into the micropores of the implanted material. Additionally, notable proliferating blood vessels were observed in the granulation tissue, these finding were in agreement with Zerbo et al. , who postulated that there is invasion of fibro-vascular tissue in the large pores of the β-TCP at early time of implantation. Moreover, our results were contradicted with that of Kondo et al.  who observed fibrous tissue around the β-TCP at early time of implantation but new bone formation was not detected and stated that bone induction did not occur until day 56 suggesting a more delayed process.
On the basis of the present study we observed from the different histological findings between these groups at 7 days interval that Nβ-TCP encouraged earlier neovascularization and enhanced bone induction in the experimental diabetic group when compared with control group. This enhancement was explained by Bignon et al. . Ohtsubo et al. , who stated that increased microporosity may provide an increased surface area for the action of angiogenic and other proteins leading to the formation of blood vessels and promoting bone induction by osteoblasts at early time points. Furthermore, Chazono et al. , suggested that micropores of β-TCP play an important role as the storage space for extracellular matrix components, including collagen, as well as providing ideal conditions for osteoinductivity.
At 28 days interval there were remarkable histological differences among the groups of the study. The control diabetic group demonstrated that at the surface of the defect there was thick band of typically organized granulation tissues and the base of the defect was almost closed by thin trabeculae of woven bone connected to the old bone forming a bridge. The decreased thickness, size and number of bone trabeculae suggestingthe retardation of the healing process in this group. These results were similar with that of Vashishth et al. , Garnero et al. , Kume et al. , who postulated that hyperglycaemia results in the accumulation of advanced glycation end products, which affects the structure of the collagen resulting in a compromised organic bone matrix quality. These advanced glycation end products may also reduce osteoblast proliferation and function and increase osteoclast-related bone resorption leading to an overall deterioration in bone quality.
Moreover, in the experimental diabetic group the defect was closed by new woven bone formation characterized by irregular bone trabeculae coalescent and anastomosed with each other in some areas forming trabecular network and harboring active bone marrow with multiple angiogenesis. This enhancement in bone healing process in compare with the control group was confirmed by the results of Curtis et al. , who investigated the long-term (in the order of days to weeks) functions of osteoblasts on nanophase ceramics and provided the first evidence of enhanced osteoblast proliferation, alkaline phosphatase synthesis, and concentration of extracellular matrix calcium on nanophase ceramics.
On the basis of this study we observed from the different histological findings between these groups at twenty eight days interval that Nβ-TCP supported bone regeneration and such observation was confirmed by Knabe et al. , who studied the effect of β-tricalcium phosphate particles with varying porosity on osteogenesis, and stated that these particles had a stimulatory effect on osteoblastic differentiation. Furthermore, it was shown that these particles attracted osteoprogenitor cells that migrated into the Interconnecting micropores of the bone substitute material and these cells differentiated into osteoblasts and thus brought about bone deposition.
| Conclusion|| |
From the scope of this investigation we can conclude the following:
- The drastic reduction to the nano-sized level of the particle of β-TCP granules could contribute to inducing both higher porosity and larger specific surface area, and it is considered that these both factors played a significantly role in the remarkable bone regeneration effects.
- Nβ-TCP induces blood vessel formation at an early stage after implantation and the blood vessels then appear to facilitate induction of osteoblasts and osteoclasts in macropores.
- Nβ-TCP has been shown to have good biocompatibility and osteoconductivity and can be used to enhance bone healing specially in retarded conditions as in DM.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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