Research Papers

Pecora 1997

Bone regeneration with a calcium sulfate barrier

Gabriele Pecora., MD, DDS.’ Sebastiano Andreana. DDS, MSe,b Joseph E. Margarone III, DDS,a Ugo Covani, MD, DDS,’ John S. Sottosanti. DDS,d Buffalo, N.Y and San Diego, Calif. DEPARTMENTS OF ORAL AND MAXILLOFACIAL SURGERY AND PERIODONTOLOGY, SUNY AT BUFFALO, NEW YORK, AND SAN DIEGO. CALIFORNIA

Objectives: Bone defects are a challenge for the dental clinician. As widely accepted in guided tissue regeneration, physically halting soft connective tissue proliferation into bone allows for bone regeneration. This concept is the “osteopromotion principle.” The aim of this study was to assess the osteopromoting effect of calcium sulfate as a barrier.

Study design. Forty male Sprague-Dawley rats were used. Mucoperiosteal flaps were raised bilaterally at buccal and lingual aspects of the mandible to expose the angles. Next, 5 mm through-and-through bony defects were created bilaterally. On the test side, sterile medical grade prehardened calcium sulfate disks were applied both lingually and buccally to cover the defect. The control side defects were left uncovered. All flaps were sutured closed. Observation times were 3, 9, 18, and 22 weeks.

Results. Histologic analysis demonstrated that at 3 weeks all test sites showed partial or complete bone healing. Similar findings were reported for all observation times. The control group showed no bone growth at 3 and 9 weeks and partial bone healing at 18 and 22 weeks.

Conclusions. This study indicates that calcium sulfate barriers can exclude connective tissues, allowing bone regeneration during healing. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1997;84:424-9)

Bone deficiencies are of major concern and affect therapies in all branches of dentistry. Bone loss in the oral cavity can be due to naturally occurring pathoses or iatrogenically induced when attempting to solve preexisting pathosis, such as periradicular bone defects.1

In the presence of an osseous defect. healing mechanisms take place with bone cells in competition with soft connective tissue cells in colonizing and filling the area. According to the principle of “osteopromotion,” connective tissue can be halted from invading the lesion,2 allowing bone cells to grow and subsequently leading to bone regeneration. This principle was elucidated by Dahlin et al.,, 3 who created mandibular through – and- through critical size defects in rats and covered the hole on one side with expanded polytetrafluoroethylene (e-PTFE) membranes. commercially known as Gore-Tex Regeneration Material barriers (Gore-Tex, W.L. Gore and Assoc.. Flagstaff, Ariz.). These were placed both buccally and lingually, leaving the opposite defect uncovered. The study showed that even 3 weeks after creation of the defects, there was visible bone growth in the test sites: bone healing in the control sites was delayed a-nd visible at longer observation times. In the latter group. dense connective tissue from the soft tissue invaded the defect in most of the samples.

Since that study, the osteopromotion technique has been used in clinical and experimental studies. 4 Barriers of different types and materials. both absorbable and nonabsorbable, have been tested and used to obtain bone regeneration. Zellin et al.’ compared 10 membranes of different materials, including ePTFE, polylactic acid, cellulose, collagen and polyglycolic acid. They demonstrated that not all absorbable and nonresorbable materials promote the same bone formation. Also, material porosity seems to affect the amount of newly formed bone.6 Despite this. however, both nonabsorbable e-PTFE membranes and absorbable polylactic/polyglycolic acid copolymers membranes do allow bone regeneration.7
Calcium sulfate (plaster of Paris) is a safe. absorbable, moldable material that has easy handling properties and reasonable cost.~ It also has been advocated and shown to allow bone regeneration in controlled clinical trials9,10 for treatment of selected dental patients 11 and in animals studies. However, contradictory results have been reported “hen calcium sulfate was used in periodontal therapy to obtain bone regeneration. An uncontrolled clinical ~tudy of 35 cases. by Alder-man 13 indicated that plaster of Paris. when placed in periodontal defects, was able to regenerate ne~k al~eolar bone. results not confirmed in a later stud~, h,,; Shaffer and App. 14

The aim of this study was to assess the effect of calcium sulfate as a barrier to alloxk bone re-eneration by assessing the osteopromotion principle.

a. Department of Oral and Maxillofacial Surgen. SUNY at Buffalo, N.Y.

b. Depariment of Pericdoiitology, SUNY at Buffalo. N.Y.

c. Cattedra di Protesi Dentaria. University of Milan. IRCCS Hosp. San Raffaele.

d. Private practice. San Diego. Calif.




Forty mate Sprague-Dawley rats weighing 450 gm were used. Permission to perform the study was given by the Laboratory Animal Facilities Committee Review at the State University of New York at Buffalo where animals were also stored for the length of the study.

The clinical protocol followed the one by Dahlin et al.3 The animals were anesthetized intraperitoneally with sodium pentobarbital. The submandibular area was shaved and disinfected with povidone-iodine solution. Mucoperiosteal flaps were raised bilaterally at buccal and lingual aspects of the mandible to expose the angles. A 5 mm diameter trephine bur on a slow speed handpiece, under constant saline solution irrigation. was used to create bilateral through-and-through bony defects. This dimension was chosen because a critical size defect implies that the defect does not heal spontaneously during the lifetime of the animal, as defined by Schmitz and Hollinger. 15 The bony disks were discarded (Fig. 1).
On the right side, prehardened medical grade sterile calcium sulfate disks (U.S. Gypsum, Medical Division. Chicago, Illinois) were applied both lingually and buccally to cover the defect (test site). The disks were custom made by mixing the calcium sulfate with sterile saline solution, poured into a mold 9 mm in diameter and 2 mm high, and sterilized in a dry heat oven at 325°F for 90 minutes. The disks were placed over the hole to cover 2 millimeters of its periphery. The disks were held by the sutured muscle tissue around the mandible (Fig.2). The left side defects (control) were left uncovered (no disk). All flaps were sutured closed with resorbable material in the muscular portion and interrupted silk 3.0 suture in the extraoral portion- Silk sutures were removed within 10 days. Four rats died during the study. Nine animals were killed at each observation time of 3, 9, 18, and 22 weeks. Animals were euthanized with an overdose of sodium pentobarbital. The mandibles were exposed and freed of soft tissues, divided into two hemimandibles. and stored in 10% buffered formalin fixative solutions. All specimens were code labeled and sent for blind histologic analysis. Samples were decalcified in DECAUSTAT solution (Decal Chemical Corp., Congers, N.Y.). Serial sections (5 ~im) were taken from the area of interest and stained with hematoxylin & cosin (H & E) and Trichrome Masson staining method, and light microscopy analysis was performed.

For evaluation. the ranking system followed the one previously described by Dahlin et al.3, and slightly modified as described in Table I. The groups were ranked in ++ (double plus), + (plus), and – (negative) according to the histologic findings and specified in Table II. The results were compared and discussed according to a descriptive analysis.



Results are summarized in Table 11. Histologic analysis demonstrated that at 3 weeks, all test sites showed partial (,6 samples) or complete bone healing (3 samples). A continuous bridge was formed between the two bone margins of the created defect (Fig. 3)



Whereas at the same observation time. the control group showed invasion of the defect by soft connective tissue (Fig. 4).
At 9 weeks, six of nine samples in the test group had c omplete bone formation and three had partial formation. In one sample, a newly formed bone mass slightly distant from the defect was observed; the defect still showed bone formation (+). In contrast. in the control group, only three samples showed partial bone formation (+), six with the defect fully filled by connective tissue (-).

A similar pattern of distribution was seen at 18 and 22 weeks. The test sites always showed more complete bone formation sites compared to the control sites (7 vs 1 and 6 vs 2. respectively, at 18 and 22 weeks). Of importance, two specimens from the control group showed complete osseous closure of the defect at 22 weeks. None of the examined samples of both groups exhibited any inflammation as demonstrated by the H & E stained specimens. Also, in none of the experimental sites were residues of the implanted material detectable at any observation time.



This study showed that the use of calcium sulfate as a barrier can halt the ingrowth of soft connective tissue, in accordance with the osteopromotion principle. As defined by Linde et al.,2 “Osteopromotion refers to the use of physical means to seal off an anatomical site in order to prevent other tissues. notably connective tissues, to interfere with osteogenesis as well as to direct bone formation.” By acting as a mechanical barrier, the calcium sulfate disks allowed bone cells to fill osseous defects. In our study. as in the study by Dahlin et al.,3 results indicate that even 3 weeks after the surgical induction of a critical size bony defect, bone regenerates, most likely because of the mechanical hindrance of a barrier to the ingrowth of connective tissue.



In the samples from our study examined 3 weeks after the procedure, none of the control group specimens showed closure of the defect, whereas in the test group, three of nine specimens had complete bridging of osseous tissue between the margins of the defect and six with ‘ partial bone healing. Only one sample showed partial bone healing in the control group. These findings were confirmed also at longer observation times in the test group, where bony defects were protected with calcium sulfate barriers. ‘Re test group at all observation times showed a higher percentage of samples with bone growth. The mechanical barrier of calcium sulfate ensured undisturbed healing to bone. preventing ingrowth of undesirable connective tissues. in accordance with the osteopromotion principle.2,4 The presence of connective tissues in areas originally occupied by bone may constitute clinical problems. such as unneeded clinical reentry after surgical endodontics 16.17 and lack of solid bone before insertion of implants. 18.19

The quality and quantity of regenerated bone is important to the outcome. In all samples at 22 weeks (Fig. 5). the bone was similar in thickness and histologic appearance to healthy rat jaws without anv surgery (pilot study). This may indicate that bone regeneration with this technique is predictable and lasts in time, ensuring long-term success.

We used calcium sulfate prehardened sterile disks as mechanical barrier. Calcium sulfate barriers have shown to allow bone defect fill in severe horizontal bone loss by Spagnuolo and Bissada .20 They used the calcium sulfate barrier to cover a composite graft consistina of demineralized freeze-dried bone and doxycycline to treat defects with horizontal bone loss. Clinical outcomes were analyzed 12 months after the operation. Defects treated in that manner showed bone vertical fill greater than the control sites. Similar results have been obtained by Garofalo et al.9 In this study the calcium sulfate barriers gave similar results compared to polylactic add in regard to the filling of angular bony defects.

Beeson 21 showed that calcium sulfate promoted bone receneration when implanted in surgically induced frontal sinus defects in dogs. All samples showed complete bone growth within 4 to 6 months. McKee and Bailey 12 created bony defects measuring 3 x 2 em at the mandibular anales and on the ramus of adult mongrel dogs. Although the periosteum was completely removed from the periphery of the defects, calcium sulfate alloplasts used to reconstruct the defects allowed bone reaeneration, even with local infections.



Because of its easy handling and moldability, calcium sulfate has always attracted the interest of clinicians in promoting bone growth; its rapid resorption 22 has been determined by Damien et al .23 to be 5 weeks. Several other materials have been tested and used in combination with plaster of Paris to reconstruct bone defects, both in orthopedics and dentistry. A composite of plaster of Paris and dextran beads was successfully used to -reconstruct calvarial bone defects in rats. 21 A composite of hydroxyapatite/plaster of Paris has been suggested for use in cranioplasty. 25

Damien et al .23 used the composite hydroxyapatitel plaster of Paris in combination with bovine osteogenic factor. This composite was able to induce complete bone fill in cranial defects. Furthermore, a study by Najar et al.26 has showed that calcium sulfate enhanced the proliferation of bone around hydroxyapatite implants. According to Parsons et al. ’27 when a composite calcium sulfatelhydroxyapatite is implanted into a defect, it shows osteoconductive properties. As the plaster of Paris is resorbed, bone is conducted from the periphery of the defect into the center, “effectively filling the space around the hydroxyapatite partiele.”23

A composite of plaster of Paris with demineralized freeze-dried bone allograft has been used successfully in extraction sites,11 surgical periodontics, 8 implant sites, 28 and in combination with guided tissue regeneration technique in endodontic surgery.

29 The latter can be of major importance in the treatment of large periapical lesions,30 where a large membrane of soft material, such as e-PTFE or polyglycolic acid, may collapse into the defect; in these cases, the plaster of Paris may serve as a space maintainer. Based on the results obtained in the present study and others,9,20 we suggest that a barrier of calcium sulfate alone can serve as membrane for guided tissue regeneration technique. This is substantiated, within the limits of the animal model adopted because bone growth was visible 3 weeks after the insertion of the disks.

Regarding resorbability of calcium sulfate, we were not able to detect any debris of material in any of the samples, probably because of the decalcifying processes (combination of EDTA and hydrochloric acid) for histologic analysis, and partially because the resorption rate already occurred at 3 weeks in the rat model.

An interesting finding resulted from the histologic analysis of a specimen from the test group at 9 weeks. A newly formed bone mass was visible, starting from one side of the created defect, extending in the muscular tissue mass of a shape resembling the disk of calcium sulfate applied as, barrier for the defect. This finding was not surprising because a study by Yamazaki et al.31 showed that when calcium sulfate with bone morphogenetic protein was inserted within muscle tissue of mice, it induced greater calcified tissue formation than when bone morphogenetic protein was implanted alone



In our case we may assume that the calcium sulfate barrier was dislodged from its site, either by the dislodging forces of the proliferating tissue or by lack of secure suture. Because of the absence of macrophages or other engulfing or digesting cells, it seems unlikely that the barrier was dislodged by these kinds of cell population. Regardless, this has been a further confirmation of the osteoconductivity property of the material tested.

Within the limits of this model, the calcium sulfate barrier lasted long enough to provide space for the osteoprogenitor cells to proliferate and heal. It may be speculated that not only by the mechanical hindrance given by the barrier used, but also the ready local availability of calcium for the mineralization of the newly formed bone have played a role in the healing, as suggested by Beeson. 21

The present investigation indirectly also confirmed the safety of plaster of Paris as bone grafting material because no inflammatory tissue was detected in the grafted area, as demonstrated by earlier studies. 12,19, 20,26,32
This study demonstrated the validity of calcium sulfate as a barrier to halt ingrowth of soft connective tissue, thereby promoting osseous formation. The safety of the tested material and its osteogenic properties12 were also observed. We suggest its use alone or in combination with other absorbable materials for the treatment of bony defects.



1. Jeffcoat M. Bone loss in the oral cavity. J Bone Miner Res 1993;S(SuppI 23):S467-73.

2. Linde A, Alberius P, Dahlin C, Bjurstam K, Sundin Y. Osteopromotion: a soft tissue exclusion principle using a membrane for bone healing and bone neogenesis. J Periodontol 1993;64(Suppl): 1116-28.

3. Dahlin C, Linde A, Gottlow J, Nyman 5. Healing of bone defects by guided tissue regeneration. Plast Reconstr Surg 1988;81:672-6.

4. Kenney EB, Jovanovic SA. Osteopromotion as an adjunct to osseointegration. Int J Prosthodont 1993;6:131-6.

5. Zellin G, Gritli-Linde A, Linde A. Healing of mandibular defects with different biodegradable and non-biodegradable membranes: an experimental study in rats. Biomaterials 1995; 16:601-9.

6. Zellin G, Linde A. Effects of different osteopromotive membrane porosities on experimental bone neogenesis in rats. Biomaterials 1996;17:695-702.

7. Sandberg E, Dahlin C, Linde A. Bone regeneration by the osteopromotion technique using bioabsorbable membrane: an experimental study in rats. J Oral Maxillofac Surg 1993;51:1100-14.

8. Sottosanti J. Calcium sulfate: a biodegradable and biocompatible barrier for guided tissue regeneration. Compend Contin Educ Dent 1992; 13:226-34.

9. Garofalo E, Bissada N, Riechetti P, Choi R Krane A, Nelson S. Composite grafts with and without absorbable barriers for the treatment of angular bony defects. J Dent Res 1996;75(Spec Issue) Abst 1995, 267.

10. Maze G, Hinkson D, Collins B. Garbin C. Bone regeneration capacity of a combination calcium sulfate-demineralized freeze dried bone allograft. J Periodontol 1994;65(10):Abst 983.

11. Sottosand L Aesthetic extractions with calcium sulfate and the principles of guided tissue regeneration. Pract Periodontics Aesthet Dent 1993;5:61-9.

12. McKee J, Bailey B. Calcium sulfate as a mandibular implant. Otolaryngol Head Neck Surg 1984;92:277-86.

13. Alderman NE. Sterile plaster of Paris as an implant in the infrahony environment: a preliminary study. J Periodontol 1969;40:11-3.

14. Shaffer CD, App GR. The use of plaster of Paris in treating infrabony periodontal defects in humans. J Periodontol 197 l;42:685-90.

15. Schmitz JP, Holllinger JO. lle critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop 1986;205:229-308.

16. Hj6rting-Hansen E, Andreasen J. Incomplete bone healing of experimental cavities in dog mandibles. Br J Oral Surg 1971;9:33-40.

17. Mascres C, Marchland J. Experimental apical scars in rats. Oral Surg Oral Med Oral Path 1980;50:164-75.

18. W einlander M. Bone growth around dental implants. Dent Clin North Am 1991;35:585-601.

19. Nyman S. Bone regeneration using the principle of guided tissue regeneration. J Clin Periodontol 199 1; 18:494-8.

20. Spagnuolo A, Bissada N. The regenerative potential of a resorbable composite barrier in the treatment of periodontitis with severe horizontal bone loss. J Dent Res 1995;74: (Special issue)Abst #685.

21. Beeson W. Plaster of Paris as an alloplastic implant in the frontal sinus. Arch Otolaryngol 198 1; 107:664-9.

22. Peltier L, Bickel E, Lillo R. Thein M. The use of plaster of Paris to fill defects in bone. Ann Surg 1957;146:61-9.

23. Damien C, Parsons J. Benedict J, W eisman D. Investigation of a hydroxyapatite and calcium sulfate composite supplemented with an osteoinductive factor. J Biomed Mater Res 1990;24:63954.

24. Snyders R, Eppley B, Krukowski M, Delftno J. Enhancement of repair in experimental calvarial bone defects using calcium sulfate and dextran beads.J Oral Maxillofac Surg 1993;51:517-24.

25. Rawlings Q Wilkins R, Hanker J, Georgiade N, Harrelson J. Evaluation in cats of a new material for cranioplasty: a composite of plaster of Paris and hydroxylapatite. 1 Neurosurg 1988;69-.269-75.

26. Nabar T, Lerdrit W, Parsons J. Enhanced osseointegration of hydroxylapatite implant material. Oral Surg Oral Med Oral Path 199 I;7E9- 15.

27. Parsons J, Ricci J, Alexander HW, Bajpai PK. Osteoconductive composite grouts for orthopedic use. Ann NY Acad Sci 1988; 523A90-207.

28. Sottosanti J. Calcium sulfate: an aid to periodontal, implant and restorative therapy. Calif Dent Assoc J 1992:20:45, 60, 62.

29. Rankow H, Krasner P. Endodontic applications of guided tissue regeneration in endodontic surgery. J Endodon 1996;22:34-43.

30. Pecora G, Kim S, Celletti R, Davarpanah M. The guided tissue regeneration in endodontic surgery: one-year postoperative results of large periapical lesions. Int Endodon J 1995;28:41-6.

31. Yamazaki Y, Oida S, Akimoto Y, Shioda S. Response of the mouse femoral muscle to an implant of a composite of bone morphogenetic protein and plaster of Paris. Clin Orthop Related Res 1988.,234:240-9.

32. Geist C, Stracher M, Grove AL Orbital augmentation by hydroxylapatite-based composites. A rabbit study and comparative analysis. Ophthalmic Plast Reconstr Surg 1991:7:8-22.