formed only into the basilar layers of the implants. The ID group showed the greatest inflammatory response as well as the greatest degree of os- teogenesis at ...
J Oral
Maxillofac
49:165-170.
Sura
1991
-
Extracranial and Mandibular Augmentation With Hydroxyapatite-Collagen in Induced Diabetic and Nondiabetic Rats MOHAMED
EL DEEB, BDS, MS,* MARK T. ROSZKOWSKl,t JOHN SANK DDS, MS,* AND IBRAHIM EL HAKIM, BDS, MS5 This study evaluated three hydroxyapatite (HA) preparations placed subperiosteally in rats given streptozotocin (70 mg/kg) to induce diabetes (ID) (n = 24) and in nondiabetic (ND) rats (n = 24) used as controls. Implants of 1) nonporous HA granules (HAG), 2) HA granules hand-mixed with bovine collagen (HACM), and 3) HA granules and purified fibrillar collagen in a preprocessed block (PFC-HA) were randomly placed in subperiosteal pockets created on the cranium and adjacent to the left/right mandibles of each rat. Six rats from each group were killed at 3, 6, 12, and 24 weeks postimplantation. Animals killed after 3 weeks showed sporadic bone proliferation and bone resorption, whereas those killed after 6, 12, and 24 weeks showed formation of new bone at the implant/bone interface. Contact of the implant with bone was a requirement for osteogenesis, but bone formed only into the basilar layers of the implants. The ID group showed the greatest inflammatory response as well as the greatest degree of osteogenesis at all intervals of time. The addition of collagen to HA appeared to reduce the inflammatory response. Specimens implanted with HACM showed the least inflammation of the three implanted materials in both ID and ND groups.
Fluctuations in serum glucose and altered carbohydrate metabolism in the edentulous diabetic patient may manifest itself as an increased loss of alveolar bone. Investigative efforts have found a gen-
eralized reduction in height of the alveolar ridge,’ reduced osteogenesis,2G3 and decreased synthesis of collagen at implanted sites4*’ Thus, the metabolic changes leading to loss of bone and impaired healing after implantation represent a further problem for the edentulous patient with diabetes who may need ridge augmentation for increased denture retention and stability. Purified bovine collagen has recently been studied as a possible matrix for holding particulate hydroxyapatite (HA) and improving its handling characteristics when it is used as an augmenting material for alveolar ridges. Mehlisch et al, in clinical trials using the HA-collagen mixture, noted that nearly all augmented ridges were firm within 3 weeks postimplantation. Waite and Matukas, using the HA-collagen mixture for zygomatic augmentation in humans, observed that no patient had clinical signs of mobility or migration of the implant at 18 months postsurgery. Animal studies using the collagen-HA mix859or tubes of collagen containing the
* Associate Professor, Division of Oral and Maxillofacial Surgery, University of Minnesota School of Dentistry, Minneapolis, MN. t Senior Dental Student, University of Minnesota School of Dentistry, Minneapolis, MN. $ Professor and Chairman, Department of Pathology, University of Maryland, Baltimore School of Dental Surgery. 5 Assistant Lecturer, Department of Oral Surgery, Azhar University School of Dentistry, Cairo, Egypt. This studv was WDDOrkd bv BRSG Grant 2SO7-RRO5322-23. NIH Training Grant DE-07098~09, and the Collagen Corporation; Palo Alto, CA. Address correspondence and reprint requests to Dr El Deeb: Department of Oral and Maxillofacial Surgery, University of Minnesota School of Dentistry, 7-174 Moos Tower, 515 Delaware St SE, Minneapolis, MN 55455. 0 1991 geons
American
Association
of Oral
and Maxillofacial
Sur-
0278-2391/91/4902-0011$3.00/O
165
HA/COLLAGEN
particles of HA,“*” generally showed a mild hosttissue inflammatory reaction to the implanted material that decreased through time. The purpose of this study was to evaluate two different preparations of collagen and HA, and HA alone, implanted subperiosteally to augment the cranium or mandible of rats with induced diabetes and nondiabetic control rats, in order to determine whether the addition of collagen to HA may affect the tissue response in rats with induced diabetes. Materials and Methods INDUCTION OF DIABETES AND PREPARATION OF ANIMALS
Forty-eight male Sprague-Dawley rats (300 to 500 g) were used in this study. Twenty-four rats received an injection of streptozotocin (70 mg/kg) in the lateral tail vein to selectively destroy pancreatic B-cells and to induce a diabetic state. The remaining 24 animals served as controls. After injection, the rats were observed for 2 weeks, during which blood glucose levels were checked with a blood glucose monitor and test strips. If the minimum level of 250 mg/dL for blood glucose was not exceeded, an additional dose of streptozotocin was administered, and the rats were monitored for an additional 2 weeks. Throughout the study, the rats were fed standard rat chow and water ad libitum. No insulin or any other medication was administered. IMPLANT PROCEDURES
After monitoring blood glucose levels in the induced diabetic group for two weeks, all 48 rats were prepared for the implant surgery. Each rat was given ketamine (10 mg/kg) and xylazine (3 mg/kg) intraperitoneally and xylocaine subcutaneously for local hemostasis at the implant sites. The predetermined sites were then shaved and aseptically prepared. Subperiosteal pockets were made in the cranial region and in the area adjacent to the right and left ramus of each mandible via extraoral incision. Equivalent volumes (1 mL) of the following HA preparations were randomly placed into each of the subperiosteal pockets: 1) nonporous HA granules (HAG; Calcitile, Calcitek, CA), 2) HA granules hand mixed with collagen in a 1:l ratio (HACM; Calcitite, Calcitek, CA, and Zyderm, Collagen Cot-p, CA), and 3) consolidated blocks of HAcollagen (PFC-HA; Alveoform Biograft, Collagen Corp, CA). Each surgical site was closed in layers with 3-O Vicryl (Ethicon, Somerville, NJ) sutures after implantation of the material. Radiographs
AUGMENTATION
IN DIABETIC RATS
were taken immediately postsurgically time each rat was killed.
and at the
HISTOLOGICPREPARATION
Six rats from each group were killed by lethal injection of sodium nembutal at 3 weeks, 6 weeks, 3 months, and 6 months postimplantation. Immediately before death, radiographs of the skull were taken of each rat. The implant and surrounding tissues were then removed in toto, immediately fixed in formalin, processed for decalcification, and embedded in paraffin. Sections of each block, 4 to 7 km thick, were prepared and replicate specimens were stained with hematoxylin and eosin (H&E) and with Masson’s trichrome stain. The samples were rated for the following tissue responses: 1) inflammation (neutrophils, lymphocytes, plasma cells, macrophages, and giant cells), 2) connective tissue response (maturity of collagen and fibroplasia), and 3) osteogenesis. Scoring methods were as follows: 0, not present; 0.5, minimal; 1.0, mild; 2.0, moderate; and 3.0, marked. Results Gross observation of the implanted sites via bimanual manipulation before the killing of each animal showed stability of the implants. All three implanted materials appeared to be firmly encapsulated by host tissues. The HAG implants showed a marked degree of settling, with displacement of particles and loss of contour. Implanted HACM showed some displacement of particles and some settling, whereas implanted PFC-HA retained most of its augmented contour in each animal (Fig 1). Radiographs taken immediately after implantation showed the PFC-HA preparation to be stable. The HACM implants showed slight displacement of
FIGURE 1. View of HACM material immediately after gross dissection of the cranial periosteum shows the cohesiveness of the implant with the host tissues.
167
ELDEEBETAL
particles and the HAG implants showed significant displacement of particles away from the implanted site. Radiographic analysis of specimens immediately before death was consistent with the gross examination. The PFC-HA implants were visibly dense and consolidated, with retention of augmented height and no displacement of particles. Conversely, the HAG and HACM implants showed settling and loss of augmented contour, with displacement of particles occurring in most specimens (Fig 2). In each specimen at each time interval, the individual granules of HA were encapsulated by bands of collagenous tissue. By the 6-month interval, the amorphous bovine collagen matrix of the HACM and PFC-HA implants had been replaced by dense, mature host connective tissue (Fig 3). At 3 weeks, 6 weeks, 3 months, and 6 months, the nondiabetic specimens showed a denser encapsulation of the HA granules in each of the three implanted materials, as compared with implants in animals with induced diabetes (Table 1). Decalcified sections from specimens after 3 weeks showed a mild inflammatory reaction consisting of macrophages and some giant cells at the HA/tissue interface in both induced diabetic and nondiabetic rats (Fig 4). The inflammatory reaction in specimens from nondiabetic animals decreased by the 6-month interval, whereas specimens from animals with induced diabetes showed a persistent inflammatory response throughout all time intervals (Table 1). Resorption of bone, as evidenced by slight osteoelastic activity at the implant/bone interface, was
FIGURE 3. At 6 months postimplantation, this control specimen shows the individual HA granules throughout the implant (x) encapsulated by dense, mature collagen (c) (Masson’s trichrome stain, original magnification x 10).
observed intermittently, and proliferation of bone was seen within some of the specimens after 3 weeks with all three implanted materials (Fig 5). At each of the subsequent time intervals, osteogenesis was seen in the most basilar portions of each of the implanted materials at the implant/bone interface (Fig 6). The nondiabetic specimens showed less growth of new bone than the specimens with induced diabetes at each time interval. When the implant was in intimate contact with the underlying bone, the basilar granules of HA were actually encased by osteoid or mature bone, and the superior portions of the implant were encapsulated by host connective tissue (Fig 7). Interestingly, the specimens from rats with induced diabetes had the greatest degree of osteogenesis at each interval when compared to the specimens from nondiabetic animals (Fig 8) (Table 1). Of the three implanted materials tested, the specimens with HAG showed the greatest degree of bone ingrowth, whereas HACM and PFC-HA specimens had little or no ingrowth. Discussion
FIGURE 2. Radiograph of nondiabetic rat at 6 months postimplantation. The PFC-HA (a) material has maintained its original contour atop the cranium, whereas the HAG (b) and HACM (c) materials have either been displaced from the initial site or have remained as a cohesive mass along the mandible.
The inflammatory reaction to the implanted materials varied between animals with induced diabetes and nondiabetic animals. Generally, the intlammatory response in nondiabetic animals decreased after 3 to 6 months. Conversely, the diabetic group showed a persistence of intlammatory cells at all intervals. Abbey et al observed a similar sustained inflammation,‘2 and in our past research with subcutaneous implants in rats with induced diabetes, we observed a similar persistent response.i3 Over-
168 Table 1.
HA/COLLAGEN
AUGMENTATION
IN DIABETIC RATS
Mean Histologic Response of Induced Diabetic and Nondiabetic Specimens HAG
Response Neutrophils Diabetic Lymphocytes Control Diabetic Plasma cells Control Diabetic Macrophages Control Diabetic Giant cells Control Diabetic Osteogenesis Control Diabetic Collagen Maturity Control Diabetic
3w
0
6W
0 .I .15 .3
0 .I
HACM 3M
0 .3
0
0 .5
0 .3 .25 .2
6M
.4 0
.3 0
3w
0 0
0 .?
0 0 0 0
0
0
0 1.3
0 1.1
.08 .7
0 0
.l .3
0 0
0 0
0
.3
0 1
0
6M
0 0
.l .7
0 0
.l
.3
0 0
.5 0
.25 1.6
.4 1.2
1.2 1.4
.7 1.4
.4 1.3
1.2 1.6
1.4 .9
1.4 1
.5 1
1.1 .7
.7 1.2
.6 0
.7 .8
1.4 1.2
1.4 .8
.03 .3
.83 .6
.16 1
0
1.3 1.2
1.3 1
.16 1.4
.25 .8
1.0 1.2
.6 1.8
2.1 1.4
.4 .?
.16 1.6
.03 .7
1.5 1
0 0
3M
.3
.67 1
1.2 1
6W
.7
1.0 1.1
.83 .l
3w
0 1
6M
.12
.27 1.2
.28 .I
3M
0 1
.7 0
6W
PFC-HA
.23 .7 1.5 1.2
1.2 1.2
1.2 1
.02 .5 1.3 1.1
.06 .8 1.7 1.2
0 1.3 1.7 1.3
1.2 .7
1.3 .7
.7
Scoring: 0, not present; 0.5, minimal; 1.0 mild; 2.0, moderate; 3.0, marked.
all, the HAG implants elicited the greatest inflammatory response of the three materials. used. Both the compacted PFC-HA and the handmixed HACM implants showed (under Masson’s trichrome stain) that the light blue, amorphous matrix of bovine collagen was gradually replaced with dense connective tissue. Bands of host fibrous tissue encapsulated the individual granules of HA in a similar fashion in each of the three implanted materials. Fibroplasia and maturity of collagen within the implanted sites were found to be similar in spec-
FIGURE 4. Control specimen (HAG) at 3 weeks postimplantation. The decalcified HA granule (x) is encapsulated by bands of host fibrous tissues next to the mandible of the animal. The inflammatory cells are observed at the HA/tissue interface and within the tissue stroma (H&E, original magnification x20).
imens from induced diabetics and from nondiabetits, in contrast to our findings in studies of implantation in soft tissue13 as well as the observation of other investigators that synthesis of collagen was markedly impaired in animals with induced diabetes. Previous investigation has supported the observation that the most active.period of osteogenesis is 3 to 4 weeks postoperatively.14 With the onset of diabetes mellitus, the normal development of bone
FIGURE 5. This 3-week specimen from a diabetic rat shows area of bone (D) in the superior portion of a PFC-HA implant along the mandible. A moderate intlammatory infiltrate is also present at the HA/tissue interface and within the loose connective tissue. Thin bands of fibrous tissue encapsulate the individual HA granules (H&E, original magnification x20).
ELDEEBETAL
169
FIGURE 6. Cranial augmentation with HACM at 3 weeks in a control animal. Immature bone (IB) can be seen infiltrating the basilar portion of the implant from the cranium (c), and the superior portions of the implant show encapsulation of the HA granules with host fibrous tissue (Masson’s trichrome stain, original magnification X 10).
FIGURE 8. Diabetic animal with mandibular HACM implant at 6 months. Note the bone infiltration (*) into the base of the implant that is in close association with the mandibular bone surface (Mn). The superior aspect of the implant is invested with host connective tissues (ct) that encapsulate the HA granules. (H&E, original magnification x 10).
has been found to be markedly decreased and the incidence of osteoporosis increased.3 In this study, specimens taken 3 weeks postoperatively from both induced diabetic and nondiabetic rats showed signs of slight resorption of bone as well as proliferation of bone at varying sites along the implant/bone interface. Silverberg” observed similar resorption of bone in a 12-week study of implantation. At 6 weeks, 3 months, and 6 months postimplantation, we observed formation of bone into the basilar portions of the implant at the implant/bone interface that was greater in specimens from diabetics compared to specimens from controls. This appears to contradict other investigative efforts that found that
the diabetic metabolic state restricts the number and activity of osteoblasts.16 Specimens from induced diabetics implanted with HAG showed the greatest ingrowth of bone, followed by specimens implanted with HA-collagen, at each interval studied. When all three materials were placed in the soft tissue of the same group of rats, no bone formation was observedi7; this indicated that neither the HA nor the composite formed by the HA/collagen mix was osteoconductive. Those animals with induced diabetes that showed the greatest inflammatory reaction had the greatest amount of osteogenesis into the implanted material. Bone was actually observed encapsulating the individual particles of HA at the basal portions of the implant. Formation of new bone did not occur when soft tissue was present between the bone and the implant. Instead, host connective tissue was observed to encapsulate the granules of HA. Thus, direct HA-to-bone contact appears necessary for osteogenesis.6*14 Further investigation is needed to determine whether bone formed within animals with induced diabetes is subject to normal remodeling because of the associated inflammatory reaction within the tissues. The overall host response to implanted HA and HA-collagen appears favorable. The bovine collagen incorporated with the particles of HA was accepted in both induced-diabetic and nondiabetic rats. Addition of collagen to the HA granules aided in maintaining superior contour of the implant. The blocks of PFC-HA maintained augmented contours throughout the 6-month course of this study. The implants of HACM had some settling, but the bo-
FIGURE 7. Six-week HACM implant in close association with the control animal’s cranium (c). Slight proliferation of bone (*) has occurred in the most basilar portions of the implant. The HA granules are primarily encapulated by fibrous tissue (H&E, original magnification X IO).
170 vine collagen retained the particles at the initial sites better than when granules of HA alone were used References 1. Saadoun AP: Diabetes and periodontal disease: A review and update. J West Sot Periodont 28: 16, 1980 (abstr) 2. McNair P, Madsbad S, Christensen MS, et al: Bone mineral loss in insulin-treated diabetes mellitus: Studies on pathogenesis. Acta Endocrinol 90:471, 1979 3. Weiss RE, Reddi AH: Influence of experimental diabetes and insulin on matrix-induced cartilage and bone differentiation. Am J Physiol 238:E200, 1980 4. Carrico TJ, Mehrhof AI, Cohen IK: Biology of wound healing. Surg Clin North Am 64:730, 1984 5. Grotendorst GR, Martin GR, Pencev DP, et al: Stimulation of granulation tissue formation by PDGF in normal and diabetic rats. J Clin Invest 76:2327, 1985 6. Mehlisch DR, Taylor TD, Leibold DG, et al: Evaluation of collagemhydroxylapatite for augmenting deticient alveolar ridges: A preliminary report. J Oral Maxillofac Surg 45:408, 1987 7. Waite PD, Matukas VJ: Zygomatic augmentation with hydroxylapatite: A preliminary report. J Oral Maxillofac Surg 44:349, 1986 8. Harvey WK, Pincock JL, Matukas VJ. et al: Evaluation of a
HA/COLLAGEN
9.
10.
11.
12.
13.
14.
IS.
16.
17.
AUGMENTATION
IN DIABETIC RATS
subcutaneously implanted hydroxylapatite-Avitene mixture in rabbits. J Oral Maxillofac Surg 43:280, 1985 Bell R, Beime OR: Effect of hydroxylapatite, tricalcium phosphate, and collagen on the healing of defects in the rat mandible. J Oral Maxiliofac Surg 46:592, 1988 Shen K, Gongloff RK: Collagen tube containers: An effective means of controlling particulate hydroxylapatite implants. J Prosthet Dent 56:68, 1986 Gongloff RK, Montgomery CK: Experimental sudy of the use of collagen tubes for implantation of particulate hydroxylapatite. J Oral Maxillofac Surg 44:847, 1985 Abbey LM, Cohen MM, Shklar G: The effect of streptozotocin-induced diabetes on the healing of artifically produced tongue wounds in rats. J Oral Surg 33:681, 1972 El Deeb M, Roszkowski MT, El Hakim I: Tissue response to hydroxylapatite in induced diabetic and nondiabetic rats: Histologic evaluation. J Oral Maxillofac Surg 48:476, 1990 Chang C, Matukas VJ, Lemons JE: Histologic study of hydroxylapatite as an implant material for mandibular augmentation. J Oral Maxillofac Surg 42:735, 1983 Silverberg M, Singh M, Sreakanth S, et al: Use of polyglycolic acid mesh to confine particulate hydroxylapatite for augmentation of bone in the rat. J Oral Maxillofac Surg 44:879, 1986 El Wahen A, El Moneim A, Ghaffar A, et al: Alveolar socket healing after extraction in normal and controlled diabetic patients. Egypt J Histol 6:206, 1983 El Deeb M. Roszkowski MT, El Hakin I. Subcutaneous implantation of hydroxyapatite/collagen in induced diabetic and nondiabetic rats. Int J Oral Maxillofac Surg 19:113, 1990
