Year : 2010 | Volume
: 21 | Issue : 1 | Page : 3--9
Osteoblast response to commercially available demineralized bone matrices - An in-vitro study
S Thanga Kumaran1, KV Arun2, Sabitha Sudarsan2, Avaneendra Talwar2, N Srinivasan3,
1 Department of Periodontics, JKK Nataraja Dental College and Hospital, Komarapalayam, India
2 Department of Periodontics, Ragas Dental College and Hospital, University of Madras, Taramani Campus, Chennai, India
3 Department of Endocrinology, Dr. A.L.M Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, India
Department of Periodontics, Ragas Dental College and Hospital, University of Madras, Taramani Campus, Chennai
Objective: Reconstruction of lost attachment apparatus is a major goal of periodontal therapy. Although various osteoinductive bone replacement grafts (BRGs) have been used with apparent clinical success, unequivocal evidence of osteoinductivity may be obtained only through the demonstration of increased osteoblastic/osteoclastic differentiation following exposure to these materials.
Materials and Methods: Bone marrow stem cells (BMSCs) obtained from rat femur were cultured in Dulbecco«SQ»s Modified Eagles Medium (DMEM) and 10% fetal bovine serum (FBS). They were then exposed to two demineralized bone matrices (DBM«SQ»s) - Grafton and Osseograft, and divided into three groups, comprising of a negative control (BMSC + DMEM + 10% FBS), Grafton, Osseograft. An osteogenic medium (OM) (10 hm dexamethasone, 10 hm b-glycerophosphate, and 50 μg/ml ascorbic acid) was added to create three subgroups comprising of a positive control (OM), Grafton with OM, Osseograft with OM.
Results: After an initial phase (up to day 5), both Grafton and Osseograft induced an increased proliferative activity in the BMSCs, which reached a plateau after day 10. These grafts also induced increased alkaline phosphatase activity when compared to the control groups and to BMSCs with an OM.
Conclusion: Both Osseograft and Grafton are capable of inducing osteoblastic proliferation and differentiation.
|How to cite this article:|
Kumaran S T, Arun K V, Sudarsan S, Talwar A, Srinivasan N. Osteoblast response to commercially available demineralized bone matrices - An in-vitro study.Indian J Dent Res 2010;21:3-9
|How to cite this URL:|
Kumaran S T, Arun K V, Sudarsan S, Talwar A, Srinivasan N. Osteoblast response to commercially available demineralized bone matrices - An in-vitro study. Indian J Dent Res [serial online] 2010 [cited 2023 Feb 9 ];21:3-9
Available from: https://www.ijdr.in/text.asp?2010/21/1/3/62796
Reconstruction of lost tissues is a major goal of periodontal therapy.  Various bone graft materials have been introduced in the last two decades in an attempt to rebuild the lost attachment apparatus. ,,, These graft materials contribute to new bone formation through osteogenic, osteoconductive or osteoinductive mechanisms.
Osteogenesis is the formation of a new bone from osteoblasts that is transplanted as a viable cellular component in autogenous bone grafts. Osteoconduction is the formation of bone where the graft acts as a scaffold, allowing the cells that form the host bone to proliferate in a favorable environment. Osteoinduction is the formation of a new bone by inducing the differentiation of undifferentiated mesenchymal cells through stimuli provided by the demineralized bone matrix. The process of osteoinduction requires the presence of a collagen/proteoglycan matrix and bioactive proteins such as bone morphogenetic proteins (BMP), neither of which is individually capable of supporting optimal bone growth. 
Evidence of osteoinduction has come from:
Animal studies, which demonstrate ectopic bone formation after subcutaneous  or intramuscular implantation  of demineralized bone matrix (DBM) in athymic mice, andIn vitro studies, which reported increased osteoblastic/osteoclastic proliferation and differentiation, following exposure to demineralized bone extract or after direct addition of BMP. These in vitro studies may not, however, simulate the conditions that exist in vertical bone defects, and in fact, there are conflicting reports on the clinical efficacy and predictability of demineralized bone matrix when used in periodontal or peri-implant osseous defects. ,,
This difference in clinical behavior has been attributed to the presence/absence of active BMP,  the bioavailability of which may be influenced by a variety of well-documented factors such as donor age, metabolic characteristics, and particle size of the graft. , The collagen/proteoglycan matrix may also exhibit considerable variation, as the commercially available DBMs do not undergo similar demineralization processes. The role of the collagen/proteoglycan matrix of the osteoinductive bone grafts in regulating osteoblastic behavior is not yet fully understood. The factors that may influence the composition of this matrix have not yet been completely elucidated.
In recent times, a new DBM (Osseograft, Advanced Biotech Products (P) Ltd. India) has been introduced, which claims to have retained the type I collagen in its native form after the demineralization process. The osseograft is said to be specially prepared from bovine cortical bone samples resulting in non-immunogenic flowable particles of approximately 250-micron size.
A truly inductive material must be capable of supporting the differentiation of uncommitted bone marrow stem cells to osteoblasts for optimal bone regeneration in periodontal or peri-implant defects. 
In this study, we have characterized the dynamics of proliferation and differentiation of bone marrow stem cells (BMSCs) when exposed to the recently introduced Osseograft and compared it to that of another DBM (Grafton, Osteotech, Inc. American Association of Tissue Banks).
Materials and Methods
Isolation of bone marrow stem cells
Bone marrow stem cells (BMSCs) were isolated according to the method described by Milne et al.  In brief, the femur was obtained from adult Wistair rats, under aseptic conditions, and the adhering muscles and connective tissues were cleaned off. The ends of the femur were cut off and the marrow thus obtained was flushed with Dulbecco's Modified Eagles Medium (DMEM) 5 ml, with 10% fetal bovine serum (FBS), and subsequently centrifuged at 1000 rpm for 10 minutes, to obtain a homogenous cell suspension. Low-density mononuclear cells were isolated and separated by density gradient Ficoll-paque  (density:1.077 g/ml). The BMSCs were then seeded in a 75 cm 2 culture flask and cultured in a humidified atmosphere (95% air, 5% CO 2 ) at 37C in DMEM containing 10 ml of penicillin/streptomycin solution and 1 ml of amphotericin B, supplemented with 10% FBS. The primary culture of BMSCs was maintained for seven days and subsequently subcultured in 12-well culture plates.
The 12-well culture plate was divided into three groups (A, B, and C) of four wells each. Each group was delineated in the manner described a little later in the text and every well was seeded with 5 x 10 4 BMSCs. [Table 1] shows the summary of the different groups.
Group A, which constituted the negative control, comprised of BMSCs cultured in DMEM with 10% FBS. Groups B and C were the experimental groups in which the bone grafts, Osseograft (Advanced Biotech products (P) Ltd. India - Joint Venture with Encoll Corp., USA) and Grafton (Osteotech, Inc. American Association of Tissue Banks), respectively, were added to the BMSCs in DMEM and FBS. Both bone grafts were obtained in their commercially available form, allowed to undergo hydrolysis in distilled water at 1 mg/ml for a period of 24 hours. Five milliliters of the supernatant solution, was then added to the culture wells.
BMSCs (5 x 10 4 cells) were added to another 12-well culture, which was split into three groups, as previously described. In addition, an osteogenic medium (OM) consisting of 10 hm dexamethasone, 10 hm b-glycerophosphate, and 50 μg/ml ascorbic acid,  was added to each of these culture wells to make subgroups I-A, I-B, and I-C. The subgroup I-A (BMSCs with osteogenic medium) was meant to act as a positive control, as the osteogenic medium has been known to have a potential to induce osteoblastic proliferation and differentiation. Subgroup I-B (Osseograft with OM) and subgroup I-C (Grafton with OM) were created to determine if any additive effect occurred from the addition of OM.
The cell proliferation and alkaline phosphatase activity (differentiation marker) was then measured in each well. Every experiment was repeated in triplicate.
The cell proliferation of the BMSCs was determined by the crystal violet bioassay as described by Wosikowsk et al.  on days 0, 2, 5, 10, and 14. In brief, the cells were washed with phosphate buffer saline (PBS) and fixed with 5% glutaraldehyde for 10 minutes. The cultures were then stained with 0.5% crystal violet in 25% methanol for 10 minutes. Subsequently, the plates were washed with water and dried. The dye taken up by the cells was extracted using 0.1 M sodium citrate. Absorbance was read at 550 nm after a suitable dilution. The optical density of each sample was then compared with the standard curve, in which the optical density was directly proportional to the viable cell number.
Assay of alkaline phosphatase
Estimation of Alkaline Phosphatase (ALPase) was carried out by the method of Andersch and Szecypinski  using p-nitro phenyl phosphate as the substrate.
The cells in each well were rinsed with PBS, harvested at eight and 14 days and processed for determining the ALPase activity, and optical density was read at 410 nm. The activity was normalized to a total cellular protein, which was determined by a protein assay using crystalline bovine serum albumin (BSA) as the standard. The ALPase activities were expressed as units of the enzyme (μmole p-nitro phenol/hour/μg protein)
For statistical analysis the mean and the SD of all the values obtained with cell culture experiments was taken and analyzed using One-way ANOVA followed by Student-Newman-Keuls test.
The results of the proliferative activity are summarized in [Table 2] and [Table 3]. On day two, a significantly higher proliferative activity was observed in group A (negative control) (P = 0.002), when compared to groups B and C. Among the subgroups, the proliferative activity was significantly higher in subgroup 1-A (positive control) (P = 0.003), when compared to subgroups I-B and I-C.The proliferative activity was significantly higher in subgroup 1-A, when compared to groups A, B, and C. There was no significant difference in the proliferative activity in the experimental groups, either between groups B and C, or subgroups I-B and I-C. Although subgroups I-B and I-C exhibited higher proliferative activity, there was statistically no significant difference in the results obtained in groups B and C [Graph 1]- [SUPPORTING:1] and [Graph 2]- [SUPPORTING:2].
On day five, the results of the proliferative activity showed the same pattern as that on day two. The proliferative activity was significantly higher in group A and subgroup I-A when compared to that on day two. There was no significant difference in the cell proliferation in the experimental groups between day five and day two.
Subsequent to day five, the experimental groups showed consistently higher proliferation when compared to the control group A and subgroup I-A.
On day 10, groups B and C (both at P = 0.006, respectively) exhibited significantly higher proliferative activity when compared to group A. Similarly, significantly lower proliferative activity was observed in subgroup I-A when compared to subgroups I-B and I-C (both at P = 0.003). Subgroup I-A exhibited a significantly higher proliferative activity when compared to group A. Between the experimental groups, however, there was no significant difference in the proliferative activity. A significantly higher proliferative activity was observed in every well on the tenth day when compared to day five.
The cell proliferative activity reached a plateau on day 10 and there was no change in any of the values obtained from any of the wells, irrespective of control or experimental, beyond this day.
Alkaline phosphatase activity
The results of ALPase activity are summarized in [Graph 3]-[SUPPORTING:3]. ALPase activity was measured on days eight and 14, as the cells were still in an actively dividing state on day eight and had ceased to do so after day 10. As expected, the ALPase activity was higher on day 14 when compared to day 10, for all groups and subgroups.
On day eight, maximum ALPase activity was seen in subgroup I-C and minimum ALPase activity was exhibited in group A. Significantly higher ALPase activity was observed in groups B (P ,, Zhang et al. reported that donor age, demineralization, and sterilization processes, might affect the bioavailability of BMP present in allografts. 
Indeed, several authors have suggested that there was no significant difference in clinical behavior when DBM was compared to osteoconductive BRGs in the vertical defects. ,, This has been attributed to the lack of active BMPs and proteoglycans/glycosaminoglycan matrix in the allografts.
There is, hence, a need for demonstration of osteoblast proliferation and differentiation when exposed to the allografts, before the material can be deemed truly inductive. A recently introduced DBM (Osseograft) claims to have retained the type I collagen matrix of the bone. There is, however, no information in the literature regarding the in vitro and in vivo behavior of this material.
Previous studies that have shown increased osteoblastic proliferation and differentiation activity following exposure to DBM, using extracts of the bone material, have established that commercially available allografts have osteoinductive potential. ,, These studies have not, however, utilized the grafts in their commercially available form, thus making it difficult to extrapolate these results to cellular behavior, when exposed to the graft particles in vertical osseous defects in clinical situations. In this study, we have used an extracted solution containing bone grafts; so as to expose the BMSCs to these materials in a manner closely approximating that of a filled defect.
Guided tissue regeneration (GTR), guided bone regeneration (GBR) and sandwich graft procedures have gained immense popularity as techniques devised to augment bone in dentulous/edentulous sites. , This has also meant that the osteoblasts exposed to the particulate bone grafts are in various stages of differentiation and hence may vary in their ability to form mineralized tissue.
In this study, we have chosen bone marrow stem cells (BMSCs) in preference to MG63 and Saos2 cell lines, because they are the earliest precursor osteoblasts. We hypothesized that a graft capable of differentiating these cells could be even more effective when exposed to committed progenitor cells. In clinical settings, the intra-marrow penetration, made prior to the graft placement would result in the exposure of BMSCs to the bone replacement graft. A subgroup I-A was created to serve as a positive control, as OM is known to influence osteoblastic behavior. It would hence serve as a benchmark from which the effects of BRG on the osteoblast may be comparatively assessed.
In the initial phase (up to day 5) proliferative activity was maximum in the subgroup I-A (Control group with OM). OM is known to regulate osteoblastic behavior, providing factors critical for osteoblastic survival and differentiation. This obviously caused increased proliferative activity in the positive controls.
In the experimental sites, proliferation was minimal during this lag phase, as osteoinductive factors such as BMPs  were known to induce differentiation of osteoblasts, resulting in retarded proliferation.
BMPs have been seen to upregulate the expression of cbfa1,  the master switch that regulates osteoblast differentiation. BMPs exert their effect primarily through the Smad pathway, although other mechanisms have been suggested. The time required for the activation of Smad and cbfa1 activation was the probable cause of the initial lag phase observed in the experimental groups.
After this lag phase, however, there was an increased proliferation of osteoblasts in groups B and C when compared to both the positive and negative controls. Once differentiated, the osteoblasts might replicate in the favorable environment created by the proteoglycans/glycosaminoglycan matrix. Maximum activity was observed in the experimental groups with addition of OM. This clearly illustrates the value of the presence of a favorable environment and the cofactors that may regulate gene expression in osteoblasts. Although maximum proliferation was observed in subgroup 1-C (Grafton with OM), there was no statistical significant difference between the two bone grafts.
ALPase activity has been used as a reliable marker for osteoblastic differentiation;  its activity may be measured in the early stages of differentiation itself. This activity was significantly higher in groups B and C when compared to the control group.
Increased ALPase activity in the experimental groups may also be explained on the basis of BMP liberation. Cbfa1 can lead to an upregulation of a variety of genes necessary for materialization in the osteoblast, such as, collagen I, OPN, BSP, and ALP genes. ,
The potent role of BMP in inducing osteoblast differentiation may be observed by the fact that groups B and C showed significantly higher ALPase activity when compared to subgroup I-A (which is by itself capable of supporting osteoblast differentiation).
Again, the ALPase was maximum in the subgroups I-B and I-C (experimental groups with the OM). The role of vitamin C in collagen proline/hydroxyproline formation, dexamethasone in stimulation of osteoblastic differentiation, and phosphates in mineralization has been well documented.
Interpretation of these results into clinical settings has to be done with care. However, within the limited scope of the study, the following conclusions may be made:
Both Grafton and Osseograft exhibit active osteoinductive potential evidenced by the increased alkaline phosphatase activity of the osteoblast.In spite of the osteoinductive potential, favorable growth characteristics increased osteoblastic proliferation and differentiation.
|1||Egelberg J. Regeneration and repair of periodontal tissues. J Periodontal Res 1987;22:233-42.|
|2||Hiatt WH, Schallhorn RG, Aaronian AJ. The induction of new bone and cementum formation. IV. Microscopic examination of the periodontium following human bone and marrow allograft, autograft and nongraft periodontal regenerative procedures. J Periodontol 1978;49;495-512. |
|3||Meffert RM, Thomas JR, Hamilton KM, Brownstein CN. Hydroxylapatite as an alloplastic graft in the treatment of human periodontal osseous defects. J Periodontol 1985;56:63-73.|
|4||Mellonig JT. Decalcified freeze-dried bone allograft as an implant material in human periodontal defects. Int J Periodontics Restorative Dent 1984;4:41-55.|
|5||Rummelhart JM, Mellonig JT, Gray JL, Towle HJ. A comparison of freeze-dried bone allograft demineralized freeze-dried bone allograft in human periodontal osseous defects. J Periodontol 1989;60:655-63. |
|6||Carranza FA, Takei HH, Cochran DL. Reconstructive periodontal surgery. In: Newman MG, Takei HH, Klokkevold PR, eds. Carranza's clinical periodontology, ed. 10. Missouri; Saunders; 2006. p. 968-90.|
|7||Reddi AH, Anderson WA. Collagenous bone matrix induced endochondral ossification and hemopoiesis. J Cell Biol 1976;69:557-72.|
|8||Guterman IA, Boman TE, Wang GJ, Balian G. Bone induction in intramuscular implants by demineralized bone matrix: Sequential changes of collagen synthesis. Coll Relat Res 1988;8:419-31.|
|9||Miyaji H, Sugaya T, Kato K, Kawamura N, Tsuji H, Kawanami M. Dentin resorption and cementum-like tissue formation by bone morphogenetic protein application. J Periodontal Res 2006;41:311-5.|
|10||Quintero G, Mellonig JT, Gambill VM, Pelleu GB Jr. A six-month clinical evaluation of decalcified freeze-dried bone allografts in periodontal osseous defects. J Periodontol 1982;53:726-30. |
|11||Richardson CR, Mellonig JT, Brunsvold MA, McDonnell HT, Cochran DL. Clinical evaluation of Bio-Oss: A bovine-derived xenograft for the treatment of periodontal osseous defects in humans. J Clin Periodontol 1999;26:421-8.|
|12||Mellonig JT. Decalcified freeze-dried bone allograft as an implant material in human periodontal defects. Int J Periodontics Restorative Dent 1984;4:41-55.|
|13||Zhang M, Powers RM Jr, Wolfinbarger L Jr. A quantitative assessment of osteoinductivity of human demineralized bone matrix. J Periodontol 1997;68:1076-84.|
|14||Syftestad G, Urist MR. Degradation of bone morphogenetic activity by pulverization. Clin Orthop Relat Res 1979;141:281-6.|
|15||Vail TB, Trotter GW, Powers BE. Equine demineralized bone matrix: Relationship between particle size and osteoinduction. Vet Surg 1994;23:386-95.|
|16||Garraway R, Young WG, Daley T, Harbrow D, Bartold PM. An assessment of the osteoinductive potential of commercial demineralized freeze-dried bone in the murine thigh muscle implantation model. J Periodontol 1998;69:1325-36.|
|17||Milne M, Kang MI, Quail JM, Baran DT. Thyroid hormone excess increases insulin-like growth factor I transcripts in bone marrow cell cultures: Divergent effects on vertebral and femoral cell cultures. Endocrinology 1998;139:2527-34.|
|18||Kveiborg M, Flyvbjerg A, Eriksen EF, Kassem M. Transforming growth factor-b 1 stimulates the production of insulin-like growth factor-I and insulin-like growth factor- binding protein-3 in human bone marrow stromal osteoblast progenitors. J Endocrinol 2001;169:549-61.|
|19||Wolfinbarger L Jr, Zheng Y. An in vitro bioassay to assess biological activity in demineralized bone. In vitro Cell Dev Biol Anim 1993;29:914-6.|
|20||Andersch MA, Szecypinski AJ. Use of p-nitro phenyl phosphate as the substrate for determination of serum acid phosphatase. Am J Clin Pathol 1947;17:571-4.|
|21||Zhang M, Powers RM Jr, Wolfinbarger L Jr. Effect(s) of the demineralization Process on the osteoinductivity of demineralized bone matrix. J Periodontol 1997;68:1085-92.|
|22||Rummelhart JM, Mellonig JT, Gray JL, Towle HJ. A comparison of freeze-dried allograft and demineralized freeze-dried bone allograft in human periodontal osseous defects. J Periodontol 1989;60:655-63. |
|23||Bowen JA, Mellonig JT, Gray JL, Towle HT. Comparison of decalcified freeze-dried bone allograft and porous particulate hydroxyapatite in human periodontal osseous defects. J Periodontol 1989;60:675-93.|
|24||Garrett S. Periodontal regeneration around natural teeth. Ann Periodontol 1996;1:621-66.|
|25||Garg AK. Augmentation and grafting of the maxillary anterior alveolar ridge. In: Garg AK, ed. Bone biology, harvesting and grafting for dental implants: Rational and clinical application. Quintessence Publishing Co, Inc.; 2004. p. 213-40.|
|26||Yamaguchi A, Komori T, Suda T. Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, Hedgehogs, and Cbfa1. Endocr Rev 2000;21:393-411.|
|27||Tou L, Quibria N, Alexander JM. Transcriptional regulation of the human RunX2/Cbfa1 gene promoter by bone morphogenetic protein-7. Mol Cell Endocrinol 2003:205:121-9.|
|28||Skillington J, Choy L, Derynck R. Bone morphogenetic protein and retinoic acid signaling cooperate to induce osteoblast differentiation of preadipocytes. J Cell Biol 2002;159:135-46.|
|29||Barnes GL, Hebert KE, Kamal M, Javed A, Einhorn TA, Lian JB, et al. Fidelity of Runx2 Activity in Breast Cancer Cells Is Required for the Generation of Metastases-Associated Osteolytic Disease. Cancer Res 2004;64:4506-13.|