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A new bio-inorganic composite as bone grafting material : in vivo study.


A bio-inorganic composite of deglued bone, chitosan, and gelatin was prepared as a substitute for autologous bone graft and evaluated in vivo using dog as experimental model. The animal experiment was evaluated by biochemical, radiography, and histo pathological studies. The serum was tested for its calcium, inorganic phosphorous and alkaline phosphatase contents at different stages of bone healing. Radiographs of experimental bone were taken at the interval of three weeks after implantation. The histopathological study of bone tissue at the defect site was carried out by euthanising the animals each at the interval of three weeks post operatively. The results had showed that there was no significant change in serum calcium and phosphorus levels during different stages fracture healing. But there was a highly significant elevation in serum alkaline phosphatase (ALP) level in all the animals up to 3rd post-operative week. Plain radiographs and photomicrographs had showed that there was no host rejection or periosteal reaction and at 9 weeks post-operative, a complete union of defect was noticed. The biomaterial developed in the laboratory, was well accepted by the host and induced bone growth.

Introduction :

Treatment of large bone defects is a continuous challenge in bone surgery. Large defects that result from trauma, infection, resection of tumours, or other causes usually do not heal spontaneously, and surgical intervention is often required. The most widely used technique for the reconstruction of a bone defect is the use of autogenous bone graft. However, the disadvantage of this technique is its limited availability and morbidity at donor site. These disadvantages led to the use allograft and xenograft. But the usage of these materials for bone repair has associated with the risk of rejection and transfer diseases. To overcome these drawbacks of endogenous and exogenous bone graft, several synthetic bone grafts have been proposed. Particularly, Hydroxyapatite (HA) is currently used world wide in practical applications as a bone substitute due to its close similarities with bone and tooth tissues [1]. HA has been used as filler for periodontal, periapical defects, alveolar ridge augmentation, and maxillofacial reconstruction [2].

We have developed a new bone grafting material containing deglued bone (DGB), chitosan, and gelatin. DGB is a byproduct of bone glue industries where glue is extracted from crushed cattle bones. Chitosan is a copolymer of 2-amino, 2-deoxy glucose and 2-acetamino, 2-deoxy glucose (chitin). It was prepared from the exoskeletaon of Pinnaeus indicus, Family--Crustaceae. Chitosan is well known for its biocompatibility, biodegradability and bioactivity [3-5]. Gelatin was used as a binder for the formulation of deglued bone and gelatin composite. It has been used in medicine as plasma expander, wound dressing adhesive and absorbent pad for surgical use. Recently, it has shown to exhibit activation macrophage and hemostatic effect [6-9].

The developed deglued bone, chitosan, and gelatin composite was analyzed for its physicochemical properties by infrared spectroscopy, X-ray diffraction and scanning electron microscopy. The results have showed that DGB has properties identical to that of HA and the composite has also retained the crystallinity [10].

In the present work, the effect of DGB, chitosan, and gelatin composite on bone healing, was studied in vivo.

Materials and Methods :

Preparation of DGB, chitosan and gelatin composite :

About 150 mg of chitosan was dissolved in 6 ml hydrochloric acid (0.1 N) at room temperature. The solution was denoted as "C". About 3 gm of gelatin was dissolved in 6 ml hydrochloric acid (0.1 N) at 55[degrees] C in a water bath. This solution was denoted as "G". The solutions C and G were mixed thoroughly at 55[degrees] C and denoted as "CG".

DGB powder was mixed with CG mixture ands made into dough. It was then filled in a glass tube diameter of 10 mm and extruded out with a glass rod. The cylindrical implants obtained were cut into required dimension and allowed curing at room temperature for 2 to 3 hours. The cured samples were dried initially at 55[degrees] C overnight and finally at 100[degrees] C for 4 hours. The dried samples were sealed in polyethylene covers and sterilized by gamma-irradiation at 2 megarods.

Experimental procedure :

The experiments were conducted on three apparently healthy dogs of either sex, weighing about 15 kgs. All the animals were operated under general anesthesia. The animals were fasted over night. Triflupromazine hydrochloride at the rate of 1 mg per kg body weight was given intramuscularly, 30 minutes before general anesthesia. Atropine sulphate at the rate 0.65 mg was given subcutaneously about 1 hour prior to operation. Anesthesia was induced with Xylazine and Ketamine at the rate of 1 mg per kg body weight. The surgery was carried out under sterile condition. The lateral aspect of the left femoral shaft was exposed and three holes of diameter about 5 mm were made, 1 cm apart using stainless steel intramedullary pin drill. Then the graft material was packed into the defect and covered by suturing the muscles in the simple continuous fashion using number one chromic catgut. The skin incision was closed by cruciate mattress pattern of sutures using braided black silk. All operations and postoperative procedures have been followed according to the Animal welfare act and the NIH guide for care and use of laboratory animals.

Evaluation of Experiments :

Biochemical Evaluation : Blood samples were collected on the pre-operative day, 5th, 21st and 42nd post-operative days. The serum was separated and tested for calcium, inorganic phosphorous and alkaline phosphatase contents.

Radiography : Radiological evaluation was done on 1st, 21st, 42nd and 63rd post-opearative days to access the graft incorporation and bone healing process. The lateral radiographs were taken with film distance of 100 cm using 50 Kv and 10 mA.

Histopathology : Animals were euthanised each at the interval of 3 weeks, post-operatively by injecting high dose Thiopentone sodium intravenously. The callus along with normal bone was collected from the fracture site. They were cut into small pieces and stored in 10% Formalin solution for 3 days and then demineralised with Formalin--Nitric acid mixture (formalin 10 ml, distilled water 8 ml, nitric acid 10 ml). The decalcified bone tissue was sectioned longitudinally, processed and embedded in paraffin. The embedded tissues were cut into 5 to 6 microns thickness and were stained with Hemotoxylin and Eosin for histological studies. The stained sections were examined under Light microscope using 100 X and 400 X magnifications.

Results & Discussion :

Biochemical evaluation :

Serum Calcium : The values of serum calcium in mg/100 ml on the pre-operative, 5th, 21st, 42nd postoperative day of animals I, II, III are shown in Table I. There was a nonsignificant reduction in serum calcium values up to 21st post-operative day in all the animals. That means there was no significant change in serum calcium level during different stages fracture healing. The decrease in the level of serum calcium could be due to increased urinary excretion after traumatic bone injury.

Serum Phosphorous : The values of serum phosphorous in mg/100 ml on the pre-operative, 5th, 21st, 42nd post-operative day of animals I, II, III are shown in Table II. A non-significant increase in serum phosphorus level was observed in all the animals up to 3 weeks post-operatively which was gradually reduced and returned back to normal values by 6th week. It was hypothesized that necrotic disintegration of the cells at the site of defect was responsible was responsible for early rise in phosphorous levels. However, the difference in serum phosphorus levels during different stages of bone healing is not significant.

Serum Alkaline Phosphatase : The values of serum alkaline phosphatase in mg/100 ml on the preoperative, 5th, 21st, 42nd post-operative day of animals I, II, III are shown in Table III. In the present study there was a highly significant elevation in serum alkaline phosphate (ALP) level in all the animals up to 3rd postoperative week and thereafter the values returned to normal by 6th post-operative week. The increase in the level of ALP following bone injury could be attributed to the proliferating osteogenic cells

Radiography : Plain radiograph, taken at the end of three weeks showed the presence of experimentally created defect in the femoral shaft with low radiographic density. Implanted material in the holes was also evident. At 6 weeks after surgery (fig. 1) slight merging of implant material with host bone was noticed as evident by more or less uniform radiographic density of bone. There was no host rejection or periosteal reaction. At 9 weeks post-operative complete union of defect was observed. The implant was well accepted by the host.


Histopathology : At the end of 3 weeks after surgery, there was immasion of fibrous tissue into the material and transformation of fibroblast to osteoblast (fig. 2). The figure shows the active bone formation. Scattered mononucleated cell infiltration and osteoblastic activity are observed at the level of defect. At the end of 6 weeks after surgery, the defect was completely filled with the woven bone. There is no evidence of implant at the level of defect. At the end of 9 weeks after surgery, osteogenic and osteolytic activities are observed with remodeling of Haversian system (fig. 3).


Conclusion : From the above results, it has been concluded that the prepared biomaterial containing DGB, chitosan and gelatin has good biocompatibility and osteogenic potential and enhance rapid bone healing by stimulating new bone ingrowth. In order to conclude the above biomaterial as ideal bone graft, further experimental and clinical studies are in progress.

References :

[1.] Cooke FW. Ceramics in Orthopedic Surgery. Clin Ortho Rel Res, 27, 135-146 (1992).

[2.] Le Geros RZ, Calcium phosphate material in restorative dentistry. A review. Adv. Dent. Res ,2, 164- 180 (1998).

[3.] Zhuang Z, Sun Q, Yang P, Lu F, Lao Z. Experimental studies on biodegradability and tissue compatibility of chitosan membrane. Shandong Yike Dahne Xue bau,34, 337-339 (1996).

[4.] Spence ML, Mccord MG. A novel composite for bone replacement. South Biomed. Engg. Conf. Proc., 16th , 257-259 (1997).

[5.] Onishi H, Machida Y. Biodegradation and distribution of water soluble chitosan in mice. Biomaterials, 20, 175-182 (1999).

[6.] Ward AG and Courts A. the Science and Technology of Gelatin, Academic Press, New York, 1997.

[7.] Tomihata K, Burczk K, Shiraki K, Ikada Y, Crosslinking and biodegradation of native and denatured collagen, American Chemical Society Symp. Series, Vol. 540, 1994.

[8.] Andersan JM, Miller, KM, Biomaterials biopcompatibility and the macrophages, Biomaterials, 5, 5- 10 (1984).

[9.] Chvapil M. Consideration on manufacturing principles of a synthetic burn dressing, A review. J Biomed Mat Res.,16, 245-263 (1982).

[10.] Saraswathy G, Pal S, Rose C, Sastry TP, A new bio-inorganic composite containing deglued bone, chitosan, and gelatin. Bulletin of Materials Science, 24, 415-420 (2001).

G. Saraswathy, T. P. Sastry, S. Pal (1), Makeena Sreenu (2), R. V. Suresh Kumar (2)

Bio-products Laboratory, Central Leather Research Institute, Adyar, Chennai-600 020.

(1) School of Bioscience and Engineering, Jadavpur University, Kolkata--700 032.

(2) Dept. of Radiology and Surgery, College of Veterinary Science, Tirupathy--517503.
Table I : Values of Serum Calcium in mg per 100 ml during different
stages of bone healing.

Period Animal I Animal II Animal III

Pre-operative day 8.7 8.7 8.3
5th day 8.5 8.6 8.3
21st day 8.4 7.6 8.0
42nd day 8.8 8.5 Nil

Period Mean [+ or -] S.D

Pre-operative day 8.56 [+ or -] 0.188
5th day 8.46 [+ or -] 0.124
21st day 8.0 [+ or -] 0.326
42nd day 8.65 [+ or -] 0.141

Table II : Values of Serum Phosphorus in mg per 100 ml during different
stages of bone healing.

Period Animal I Animal II Animal III

Pre-operative day 4.0 5.5 4.8
5th day 4.4 5.8 5.3
21st day 5.4 6.3 5.0
42nd day 4.2 5.6 Nil

Period Mean [+ or -] S.D

Pre-operative day 4.77 [+ or -] 0.613
5th day 5.16 [+ or -] 0.579
21st day 5.56 [+ or -] 0.543
42nd day 4.9 [+ or -] 0.7

Table III : Values of Serum alkaline phosphatase in IU/L during
different stages of bone healing.

Period Animal I Animal II Animal III

Pre-operative day 51.0 70.0 80.0
5th day 153.0 180.0 233.0
21st day 160.0 192.0 245.0
42nd day 55.0 65.0 Nil

Period Mean [+ or -] S.D

Pre-operative day 67.0 [+ or -] 12.02
5th day 188.6 [+ or -] 33.23
21st day 199.0 [+ or -] 35.05
42nd day 60.0 [+ or -] 5.0
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Article Details
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Author:Saraswathy, G.; Sastry, T.P.; Pal, S.; Sreenu, Makeena; Kumar, R.V. Suresh
Publication:Trends in Biomaterials and Artificial Organs
Geographic Code:9INDI
Date:Jan 1, 2004
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