A COMPARISON IN HEALING OF SKULL DEFECT REPAIRED WITH FOUR DIFFERENT KINDS OF GRAFT MATERIALS IN RABBITS
作者：DING Zhen-qi(丁真奇)1, TAN Fu-sheng(谭富生)2, WU Zu-yao(吴祖尧)2
单位：TAN Fu-sheng(谭富生)2, WU Zu-yao(吴祖尧)2 Department of Orthopedic Surgery 1st Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400042
关键词：
中华创伤杂志980357 Objective To investigate the difference in human deproteinized bone (h-DPB), composite of h-DPB and bone morphogenetic protein (h-DPB/BMP), composite of hydroxyapatite (HAP/BMP) and hydroxyapatite (HAP) in repairing rabbit skull bone defects.
, 百拇医药
Methods Seventy-eight adult white rabbits were equally divided into A and B groups at random. The experimental models were made by trephining a circular defects 1cm in diameter on both parietal bones of each rabbits. Bone defects in each rabbit were repaired with one h-DPB/BMP and one HAP/BMP in group A, with one h-DPB and one HAP grafts in group B. The 35 S,45Ca contents and ash weight of the implants were measured and their roentgenological and histological changes were observed.
, 百拇医药
Results The osteogenesis of the graft materials was h-DPB/BMP>HAP/BMP (P<0.05)>h-DPB and HAP (P<0.01). No statistical difference was found between h-DPB and HAP.
Conclusion Deproteinized bones serve as a better carrier for BMP than HAP, and is worthy to be recommended for clinical use.
Bone defects are usually repaired by transplanting deproteinized bone (DPB), hydroxyapatite (HAP) and their composites. However, few reports have been found in literature on quantitating the effect of the transplant in a skeletal defect and which is better carrier for bone morphogenetic protein (BMP). We utilized human DPB(h-DPB), h-DPB/BMP, HAP/BMP and HAP to repair rabbits skull defects and analyse quantitatively their abilities of osteogenesis.
, http://www.100md.com
MATERIALS AND METHODS
Fresh human femur or tibial metaphyseal bones obtained from traumatic amputation were deproteinized in 20% hydrogen peroxide for 72 hours at 37°. Then fat was extracted with ether for 48 hours. A dense microporous h-DPB was made in the same way. The collagen stain of h-DPB demonstrated that h-DPB contained some collagen fibers. X-ray diffraction showed the molecular formula of h-DPB was Ca5 (PO4)3(OH). Large blocks of h-DPB were milled into 0.2 cm thickness and cut into circular blocks 1cm in diameter. Seventy-eight circular blocks, each weighing 73mg, were selected and equally divided into group A and group B at random.
, 百拇医药
HAP granules weighing 5694mg, provided by Beijing Aeronautical Material Research Institute, were equally divided into group A and group B. HAP granules in group B were divided into 39 portions each weighing 73mg.
Porcine BMP-PI was obtained using the method of Wu et al.1 BMP was combined with h-DPB and HAP separately in group A. HAP in group A was divided into 39 portions. Each h-DPB contained 2.8-3.3mg BMP, the amount of which combined with h-DPB and HAP showed no difference of significance (P>0.05).
, 百拇医药
Seventy-eight large white rabbits weighing 2.53kg were equally randomized into groups A and B. The experimental models were made by trephining a circular defect 1cm in diameter in both parietal bones of each rabbit. Each rabbit received one h-DPB/BMP and a portion of HAP/BMP in group A, one h-DPB and a portion of HAP on the left and right sides respectively in group B.
The discs of parietal bone removed were ashed. The ash weight of the discs between group A and group B showed no difference of statistical significance.
, http://www.100md.com
One, two, four and six weeks after implantation, 2 rabbits from each group were sacrificed and examined histologically.
By six weeks, all rabbits were sacrificed for roentgenographical examination.
On 9th day, 13 rabbits in group A and 12 in group B were given 35 S intravenously at a dose of 12.5 μ Ci/kg body weight. Twelve hours after injection, implants 1cm in diameter were removed by trephination. The samples were prepared by the method of Reddl et al.2 On 39th day all rabbits were given 45 Ca intravenously at a dose of 12.5 μCi/kg body weight. Three days after injection the rabbits were sacrificed and the implants were removed by trephine and the parietals of similar size around the bone defects were removed. All samples were ashed in a Muffle furnace at 800° and weighed for determination of radioactivity.
, http://www.100md.com
RESULTS
Three rabbits in group A and 4 in group B died. These animals were excluded from the study.
One week after implantation, many capillaries fibroblasts, mesenchymal cells and a few inflammatory cells were observed in all grafts. In group B neither bone nor cartilage formation was observed. However, in group A chondrocytes and osteoblasts in grafts were seen. By two weeks all grafts showed hypertrophic chondrocytes and weave bone formation. By four weeks irregular lamellar bone were observed in both groups; in six weeks new bone formation increased in both groups. h-DPB was obviously absorbed, while HAP was only slightly absorbed. At each stage, the area of new bone in group B was smaller than that in group A and the central portion of the grafts in group A showed cartialage and new bone formation (Figs 1～4).
, http://www.100md.com
Fig 1 One week after implantation of human deproteinzed bone (h-DPB). A great number of mesenchymal cells appear in the grafted bone (GB). HE10×10
Fig 2 One week after implantation of h-DPB/BMP. cartilage (c) formation appears in the bone graft (GB). Osteoblasts range along the cartilage periphery. HE 20×10
Fig 3 Six weeks after implantation of h-DPB. Obvious resorption in bone graft. New bone (NB) embraces bone graft (GB). HE10×10
, http://www.100md.com
Fig 4 Six weeks after implantation of h-DPB/BMP. New bone (NB) is increasing and “nibbling” bone graft (GB). HE20×20
On roentegenographs h-DPB/BMP grafts were almost completely incorporated by new bone, in HAP/BMP grafts, a large portion were incorporated by new bone, whereas in group B bone defects were only partly repaired. (Figs 5 and 6). The results of measurement of 35 S,45 Ca and ash weight are shown in Tables 1 and 2.
, 百拇医药
Fig 5 Six weeks after implantation of h-DPB(right) and HAP (left). The density of h-DPB and HAP is increasing. Bone defects are partly repaired by new bone.
Fig 6 Six weeks after implantation of h-DPB/BMP (right) and HAP/BMP(left). The h-DPB/BMP grafts are almost completely incorporated by new bone. The HAP/BMP grafts are mostly incorporated by new bone.
Table 1 35 S and 45 Ca incorporation and ash weight of the four graft materials implanted into rabbit skull defect Type
, http://www.100md.com
n
35 Sincorporation at day 10
n
Day 42
Acid soluble (Bq/sample)
Acid insoluble (Bq/sample)
45 Ca incorporation(Bq/sample)
Ash weight (mg/sample)
h-DPB/BMP
13
, 百拇医药
5709.48±432.09
503.9±72.69
15
7388.31±885.84
71.70±11.02
HAP/BMP
13
5401.91±605.46
435.26±82.84
15
6779.96±635.30
88.24±12.06
, 百拇医药
H-DPB
12
4714.78±441.59
293.72±55.34
15
5335.07±478.92
60.91±9.50
HAP
12
1780.78±416.09
274.39±50.01
15
, 百拇医药
5272.72±434.09
79.30±10.54
1. 35 S and 45 Ca incorporation: h-DPB/BMP>HAP/BMP (P>0.05)>h-DPB and HAP (P<0.01). No differences were found between h-DPB and HAP (P>0.05
2. Ash weight: h-DPB/BMP>h-DPB(P<0.01), HAP/BMP>HAP (P<0.01)Table 2 The ash weight and 45 Ca incorporation of parietals around the defects in rabbits Group
n
, http://www.100md.com
Ash weight
(mg/sample)
45 Ca incorporation
(Bq/sample)
A
Left
15
88.02±16.68
11176.40±2537.17
right
15
87.73±15.96
, 百拇医药
11307.27±2295.12
B
Left
15
89.20±17.01
11283.09±2410.49
right
15
88.48±16.59
11351.93±2365.48
No difference in the same group (P>0.05)
, 百拇医药
No difference between A and B groups (P>0.05)
DISCUSSION
The quantitative analysis of new bone formation in orthotopic grafting sites can directly reflect the ability of grafts in healing bone defects, but it is easily affected by other factors. In this study four comparable grafts and discs from all skull defect models were subjected to statistical analysis (P>0.05), therefore there was much consistency in the experiment conditions.
, 百拇医药
Many methods have been used in study of callus. Measuring the amount of minerals has been used successfully for this purpose.3，4 It can fairly reflect the difference of the content of callus. But if the content of callus in each stage can be reflected, large amount of samples are needed. Because this experiment is restricted by the amount of sample. The amount of minerals in the implants were only measured at days 10 and 42 respectively, when cartilage formation and calcification were at peak,5 But the results also showed the difference when h-DPB/BMP, HAP/BMP, h-DPB and HAP were used in repairing rabbits skull defects.
, 百拇医药
Induction of osteogenesis is the main purpose of any bone grafting procedure. Bone induction is attributed to BMP. Other bone growth factors may participate the mitogenic and differentiative activities of osteogenic cells induced by BMP.6 Two compostie grafts having both osteoinduction and osteoconduction made by BMP to combine separately with h-DPB and HAP are far superior to both h-DPB and HAP which have only osteoconduction in repairing bone defect.
, 百拇医药 Treatment with hydrogen peroxide might reduce the antigenic properties of the proteins in xenogeneic bone by oxidizing the side chains in the amino acids.7 After h-DPB was implanted into rabbits skull defects, the wounds of host showed no evidence of obvious inflammatory response. Cartilage and new bone formation were found in h-DPB at two weeks. The host did not suffer from obvious rejection against h-DPB. At six weeks after implantation, because h-DPB were obviously resorbed and a large amount of new bone was deposited, h-DPB was replaced easily by host bone.
, 百拇医药
As compared with HAP, h-DPB has more merits because (1) h-DPB similar to host's bone matrix, contains natural dense microporous structure and collagen fibers, which serves as better a carrier of BMP. (2) h-DPB has an elastic property, so that it is easily modelled into various shapes to fit into the bone defect of particular size. (3) h-DPB is easily resorbed and replaced by newly formed bone in the body of the host.
DING Zhen-qi(丁真奇) Department of Traumatic Surgery, 175th Hospital of PLA Zhangzhou 363000,REFERENCES
, 百拇医药
[1] Wu ZY, Hu XB. Separation and purification of porcine bone morphogenetic protein. Clin Orthop 1988; 23∶239.
[2] Reddi AH, Shllivan NE. Matrix-induced endochondral bone differentiation. Endocrinology 1980; 107∶1291.
[3] Gao TJ, Tan FS. Promoting effect of L-Dopa on experimental fracture healing. Chin J of Surg 1990; 28∶367.
[4] Aspenbery P, Wittbier J, Thorngren KG. Pulverized bone matrix as an injectable bone graft in rabbit radius defects. Clin Orthop 1986; 206∶2761.
, 百拇医药
[5] Chen QX, Yuan ZX, Liu J, et al. The relationship between proliferation of bone repairing cells and bone formation during fracture healing-An experimental study. Chin J Orthop 1992; 12∶290.
[6] Ding ZQ, Tan FS, Wu ZY. A survey of bone growth factors. Chongqing Med J 1993; 22∶115.
[7] Quan ZX, Wu ZY, Zhu DY. The observation of immune response to porcine bone morphogenetic (p-BMP) in rabbits. Chin J Trauma 1991; 7∶139., 百拇医药(DING Zhen-qi(丁真奇)1, TAN Fu-sheng(谭富生)2, WU Zu-yao(吴祖尧)2)
单位：TAN Fu-sheng(谭富生)2, WU Zu-yao(吴祖尧)2 Department of Orthopedic Surgery 1st Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400042
关键词：
中华创伤杂志980357 Objective To investigate the difference in human deproteinized bone (h-DPB), composite of h-DPB and bone morphogenetic protein (h-DPB/BMP), composite of hydroxyapatite (HAP/BMP) and hydroxyapatite (HAP) in repairing rabbit skull bone defects.
, 百拇医药
Methods Seventy-eight adult white rabbits were equally divided into A and B groups at random. The experimental models were made by trephining a circular defects 1cm in diameter on both parietal bones of each rabbits. Bone defects in each rabbit were repaired with one h-DPB/BMP and one HAP/BMP in group A, with one h-DPB and one HAP grafts in group B. The 35 S,45Ca contents and ash weight of the implants were measured and their roentgenological and histological changes were observed.
, 百拇医药
Results The osteogenesis of the graft materials was h-DPB/BMP>HAP/BMP (P<0.05)>h-DPB and HAP (P<0.01). No statistical difference was found between h-DPB and HAP.
Conclusion Deproteinized bones serve as a better carrier for BMP than HAP, and is worthy to be recommended for clinical use.
Bone defects are usually repaired by transplanting deproteinized bone (DPB), hydroxyapatite (HAP) and their composites. However, few reports have been found in literature on quantitating the effect of the transplant in a skeletal defect and which is better carrier for bone morphogenetic protein (BMP). We utilized human DPB(h-DPB), h-DPB/BMP, HAP/BMP and HAP to repair rabbits skull defects and analyse quantitatively their abilities of osteogenesis.
, http://www.100md.com
MATERIALS AND METHODS
Fresh human femur or tibial metaphyseal bones obtained from traumatic amputation were deproteinized in 20% hydrogen peroxide for 72 hours at 37°. Then fat was extracted with ether for 48 hours. A dense microporous h-DPB was made in the same way. The collagen stain of h-DPB demonstrated that h-DPB contained some collagen fibers. X-ray diffraction showed the molecular formula of h-DPB was Ca5 (PO4)3(OH). Large blocks of h-DPB were milled into 0.2 cm thickness and cut into circular blocks 1cm in diameter. Seventy-eight circular blocks, each weighing 73mg, were selected and equally divided into group A and group B at random.
, 百拇医药
HAP granules weighing 5694mg, provided by Beijing Aeronautical Material Research Institute, were equally divided into group A and group B. HAP granules in group B were divided into 39 portions each weighing 73mg.
Porcine BMP-PI was obtained using the method of Wu et al.1 BMP was combined with h-DPB and HAP separately in group A. HAP in group A was divided into 39 portions. Each h-DPB contained 2.8-3.3mg BMP, the amount of which combined with h-DPB and HAP showed no difference of significance (P>0.05).
, 百拇医药
Seventy-eight large white rabbits weighing 2.53kg were equally randomized into groups A and B. The experimental models were made by trephining a circular defect 1cm in diameter in both parietal bones of each rabbit. Each rabbit received one h-DPB/BMP and a portion of HAP/BMP in group A, one h-DPB and a portion of HAP on the left and right sides respectively in group B.
The discs of parietal bone removed were ashed. The ash weight of the discs between group A and group B showed no difference of statistical significance.
, http://www.100md.com
One, two, four and six weeks after implantation, 2 rabbits from each group were sacrificed and examined histologically.
By six weeks, all rabbits were sacrificed for roentgenographical examination.
On 9th day, 13 rabbits in group A and 12 in group B were given 35 S intravenously at a dose of 12.5 μ Ci/kg body weight. Twelve hours after injection, implants 1cm in diameter were removed by trephination. The samples were prepared by the method of Reddl et al.2 On 39th day all rabbits were given 45 Ca intravenously at a dose of 12.5 μCi/kg body weight. Three days after injection the rabbits were sacrificed and the implants were removed by trephine and the parietals of similar size around the bone defects were removed. All samples were ashed in a Muffle furnace at 800° and weighed for determination of radioactivity.
, http://www.100md.com
RESULTS
Three rabbits in group A and 4 in group B died. These animals were excluded from the study.
One week after implantation, many capillaries fibroblasts, mesenchymal cells and a few inflammatory cells were observed in all grafts. In group B neither bone nor cartilage formation was observed. However, in group A chondrocytes and osteoblasts in grafts were seen. By two weeks all grafts showed hypertrophic chondrocytes and weave bone formation. By four weeks irregular lamellar bone were observed in both groups; in six weeks new bone formation increased in both groups. h-DPB was obviously absorbed, while HAP was only slightly absorbed. At each stage, the area of new bone in group B was smaller than that in group A and the central portion of the grafts in group A showed cartialage and new bone formation (Figs 1～4).
, http://www.100md.com
Fig 1 One week after implantation of human deproteinzed bone (h-DPB). A great number of mesenchymal cells appear in the grafted bone (GB). HE10×10
Fig 2 One week after implantation of h-DPB/BMP. cartilage (c) formation appears in the bone graft (GB). Osteoblasts range along the cartilage periphery. HE 20×10
Fig 3 Six weeks after implantation of h-DPB. Obvious resorption in bone graft. New bone (NB) embraces bone graft (GB). HE10×10
, http://www.100md.com
Fig 4 Six weeks after implantation of h-DPB/BMP. New bone (NB) is increasing and “nibbling” bone graft (GB). HE20×20
On roentegenographs h-DPB/BMP grafts were almost completely incorporated by new bone, in HAP/BMP grafts, a large portion were incorporated by new bone, whereas in group B bone defects were only partly repaired. (Figs 5 and 6). The results of measurement of 35 S,45 Ca and ash weight are shown in Tables 1 and 2.
, 百拇医药
Fig 5 Six weeks after implantation of h-DPB(right) and HAP (left). The density of h-DPB and HAP is increasing. Bone defects are partly repaired by new bone.
Fig 6 Six weeks after implantation of h-DPB/BMP (right) and HAP/BMP(left). The h-DPB/BMP grafts are almost completely incorporated by new bone. The HAP/BMP grafts are mostly incorporated by new bone.
Table 1 35 S and 45 Ca incorporation and ash weight of the four graft materials implanted into rabbit skull defect Type
, http://www.100md.com
n
35 Sincorporation at day 10
n
Day 42
Acid soluble (Bq/sample)
Acid insoluble (Bq/sample)
45 Ca incorporation(Bq/sample)
Ash weight (mg/sample)
h-DPB/BMP
13
, 百拇医药
5709.48±432.09
503.9±72.69
15
7388.31±885.84
71.70±11.02
HAP/BMP
13
5401.91±605.46
435.26±82.84
15
6779.96±635.30
88.24±12.06
, 百拇医药
H-DPB
12
4714.78±441.59
293.72±55.34
15
5335.07±478.92
60.91±9.50
HAP
12
1780.78±416.09
274.39±50.01
15
, 百拇医药
5272.72±434.09
79.30±10.54
1. 35 S and 45 Ca incorporation: h-DPB/BMP>HAP/BMP (P>0.05)>h-DPB and HAP (P<0.01). No differences were found between h-DPB and HAP (P>0.05
2. Ash weight: h-DPB/BMP>h-DPB(P<0.01), HAP/BMP>HAP (P<0.01)Table 2 The ash weight and 45 Ca incorporation of parietals around the defects in rabbits Group
n
, http://www.100md.com
Ash weight
(mg/sample)
45 Ca incorporation
(Bq/sample)
A
Left
15
88.02±16.68
11176.40±2537.17
right
15
87.73±15.96
, 百拇医药
11307.27±2295.12
B
Left
15
89.20±17.01
11283.09±2410.49
right
15
88.48±16.59
11351.93±2365.48
No difference in the same group (P>0.05)
, 百拇医药
No difference between A and B groups (P>0.05)
DISCUSSION
The quantitative analysis of new bone formation in orthotopic grafting sites can directly reflect the ability of grafts in healing bone defects, but it is easily affected by other factors. In this study four comparable grafts and discs from all skull defect models were subjected to statistical analysis (P>0.05), therefore there was much consistency in the experiment conditions.
, 百拇医药
Many methods have been used in study of callus. Measuring the amount of minerals has been used successfully for this purpose.3，4 It can fairly reflect the difference of the content of callus. But if the content of callus in each stage can be reflected, large amount of samples are needed. Because this experiment is restricted by the amount of sample. The amount of minerals in the implants were only measured at days 10 and 42 respectively, when cartilage formation and calcification were at peak,5 But the results also showed the difference when h-DPB/BMP, HAP/BMP, h-DPB and HAP were used in repairing rabbits skull defects.
, 百拇医药
Induction of osteogenesis is the main purpose of any bone grafting procedure. Bone induction is attributed to BMP. Other bone growth factors may participate the mitogenic and differentiative activities of osteogenic cells induced by BMP.6 Two compostie grafts having both osteoinduction and osteoconduction made by BMP to combine separately with h-DPB and HAP are far superior to both h-DPB and HAP which have only osteoconduction in repairing bone defect.
, 百拇医药 Treatment with hydrogen peroxide might reduce the antigenic properties of the proteins in xenogeneic bone by oxidizing the side chains in the amino acids.7 After h-DPB was implanted into rabbits skull defects, the wounds of host showed no evidence of obvious inflammatory response. Cartilage and new bone formation were found in h-DPB at two weeks. The host did not suffer from obvious rejection against h-DPB. At six weeks after implantation, because h-DPB were obviously resorbed and a large amount of new bone was deposited, h-DPB was replaced easily by host bone.
, 百拇医药
As compared with HAP, h-DPB has more merits because (1) h-DPB similar to host's bone matrix, contains natural dense microporous structure and collagen fibers, which serves as better a carrier of BMP. (2) h-DPB has an elastic property, so that it is easily modelled into various shapes to fit into the bone defect of particular size. (3) h-DPB is easily resorbed and replaced by newly formed bone in the body of the host.
DING Zhen-qi(丁真奇) Department of Traumatic Surgery, 175th Hospital of PLA Zhangzhou 363000,REFERENCES
, 百拇医药
[1] Wu ZY, Hu XB. Separation and purification of porcine bone morphogenetic protein. Clin Orthop 1988; 23∶239.
[2] Reddi AH, Shllivan NE. Matrix-induced endochondral bone differentiation. Endocrinology 1980; 107∶1291.
[3] Gao TJ, Tan FS. Promoting effect of L-Dopa on experimental fracture healing. Chin J of Surg 1990; 28∶367.
[4] Aspenbery P, Wittbier J, Thorngren KG. Pulverized bone matrix as an injectable bone graft in rabbit radius defects. Clin Orthop 1986; 206∶2761.
, 百拇医药
[5] Chen QX, Yuan ZX, Liu J, et al. The relationship between proliferation of bone repairing cells and bone formation during fracture healing-An experimental study. Chin J Orthop 1992; 12∶290.
[6] Ding ZQ, Tan FS, Wu ZY. A survey of bone growth factors. Chongqing Med J 1993; 22∶115.
[7] Quan ZX, Wu ZY, Zhu DY. The observation of immune response to porcine bone morphogenetic (p-BMP) in rabbits. Chin J Trauma 1991; 7∶139., 百拇医药(DING Zhen-qi(丁真奇)1, TAN Fu-sheng(谭富生)2, WU Zu-yao(吴祖尧)2)