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ORIGINAL RESEARCH Table of Contents   
Year : 2009  |  Volume : 20  |  Issue : 1  |  Page : 7-12
Osteoblast response (initial adhesion and alkaline phosphatase activity) following exposure to a barrier membrane/enamel matrix derivative combination


Department of Periodontics and Implantology, Ragas Dental College and Hospital, Chennai, India

Click here for correspondence address and email

Date of Submission27-Nov-2007
Date of Decision04-Jul-2008
Date of Acceptance23-Jul-2008
 

   Abstract 

Background and Objective: The enamel matrix derivative (EMD) has been used in combination with barrier membranes to optimize regeneration in vertical osseous defects. However, the osteoblast response when exposed to the EMD/barrier membrane combination has not yet been evaluated. The osteoblast behavior when exposed to a combination of regenerative materials must be evaluated to fully understand their effect on bone regeneration. Therefore, the present study was undertaken to estimate the initial adhesion and alkaline phosphatase (ALP) activity of an osteoblast cell line (SaOS-2) when exposed to four commercially available resorbable membranes and determine if the addition of EMD had any modulatory effect on osteoblast behavior.
Materials and Methods: 5 x 104 SaOS-2 cells between passages 7-10 were cultured in two 24-well culture plates. Plate A was used for the adhesion assay and Plate B was used for the ALP assay. A MTT (3-[4, 5-dimethylthiazolyl-2]-2, 5-diphenyltetrazolium bromide) assay was done after 24 hours to determine the adhesion of the osteoblastic cells to four barrier membranes: 1) a non cross-linked porcine Type I and III collagen membrane (BG), 2) a weakly cross-linked Type I collagen membrane (HG), 3) a glutaraldehyde cross-linked bovine Type I collagen (BM), and 4) a resorbable polymer membrane (CP). Osteoblast differentiation was studied using an ALP assay with p-nitro phenyl phosphate as the substrate at 24 hours, 72 hours, and 1 week. A total of 50 g/ml of EMD dissolved in 10 mM acetic acid was added into each well and the entire experimental protocol outlined above was repeated.
Results: The osteoblast adhesion to collagen barriers showed a statistically insignificant reduction following the addition of EMD. Adhesion to the polymer barrier, although significantly lower when compared with collagen barriers, was unaffected by the addition of EMD. ALP activity after 1 week among the various groups was as follows:
EMD alone (75.592.5)>EMD/BG(64.783.04)>EMD/HG(55.403.89)≈EMD/BM(54.754.17)>BG (51.322.76)>HG(49.922.4)>BM(48.141.4)>Control(46.291.39)>EMD/CP (37.463.54)>CP(32.121.49)
Conclusion: There was no additive effect on osteoblast adhesion/ALP activity following exposure to an EMD/polymer combination. EMD/collagen positively influences osteoblast differentiation in a time dependent manner.

Keywords: Adhesion/differentiation, barrier membranes, enamel matrix derivative, osteoblast

How to cite this article:
Thangakumaran S, Sudarsan S, Arun K V, Talwar A, James JR. Osteoblast response (initial adhesion and alkaline phosphatase activity) following exposure to a barrier membrane/enamel matrix derivative combination. Indian J Dent Res 2009;20:7-12

How to cite this URL:
Thangakumaran S, Sudarsan S, Arun K V, Talwar A, James JR. Osteoblast response (initial adhesion and alkaline phosphatase activity) following exposure to a barrier membrane/enamel matrix derivative combination. Indian J Dent Res [serial online] 2009 [cited 2019 Feb 17];20:7-12. Available from: http://www.ijdr.in/text.asp?2009/20/1/7/49048
The major goals of periodontal therapy are the regeneration of lost attachment apparatus and preventing disease progression. [1] Regeneration is defined as the reproduction or reconstitution of a lost or injured part. [2] Guided tissue regeneration (GTR) utilizes physical barriers to exclude unwanted cells from the wound space to promote periodontal regeneration. [3],[4] In addition to providing cell occlusivity, space maintenance, tissue integration, and biocompatibility, these barrier membranes must, ideally, possess the ability to induce cellular proliferation and differentiation. [5] Adhesion to a substrate is an initial pre-requisite for cells before they proceed further into their proliferation and differentiation cascade. [6] Mesenchymal cell adhesion to barrier membranes is dependent on the type and nature of the barrier used and is thought to positively influence regenerative outcomes. [7],[8]

True regeneration, however, remains elusive following GTR therapy as the regenerated cementum was predominantly cellular without functionally oriented periodontal fibers. [9] In recent years, regenerative therapy has utilized the biomimetic approach, i.e., replication of events occurring during tissue morphogenesis in the periodontal wound space. Several authors have used an enamel matrix derivative (EMD; Emdogain, Straumann Biologics, Waltham, MA) in regenerative periodontal therapy and reported an enhanced defect fill and acellular cementum formation. [10],[11],[12] EMD's ability to induce osteoblast proliferation and differentiation has been well documented. [13]

The physical properties of the EMD gel are such that adequate space maintenance is difficult to achieve. In an effort to overcome this limitation, EMD has been used in combination with several bone replacement grafts and barrier membranes. [14],[15],[16],[17] The barrier membrane is thought to aid in cell exclusion while an EMD would stimulate the differentiation of the mesenchymal progenitor cells in the wound space. However, the exact nature of osteoblast behavior, when exposed to a combination of EMD and barrier membrane, is yet to be fully elucidated. The success of such therapeutic modalities depends on the ability of materials to act synergistically to promote osteoblast activity. Two important osteoblast characteristics, adhesion and alkaline phosphatase (ALP) activity, play an important role in the initial steps towards formation of new bone. The aim of this study was to evaluate the initial response (adhesion and ALP activity) of osteoblasts (SaOS-2) to four resorbable membranes and to determine if the addition of EMD would affect these characteristics.


   Materials and Methods Top


Regenerative Materials Examined

The four commercially available GTR membranes with different composition and structure examined are as follows:

  • BG: Non cross-linked porcine Types I and III collagen membrane (BioGuide, Geistlich Biomaterials, Wolhusen, Switzerland)
  • BM: Glutaraldehyde cross-linked bovine Type I collagen (BioMend, Sultzer Calciteck Inc., Carlsbad, CA, USA)
  • HG: A Type I collagen membrane (Healiguide, Advanced Biotech Products (P) Ltd., Encoll Corp., Fremont, CA, USA)
  • CP: A resorbable polymer membrane (Cytoplast, Osteogenics Biomedical Inc., Lubbock, TX, USA.)
Cell Culture

A SaOS-2 cell line was procured from the National Center for Cell Sciences (NCCS), Pune, India. The cells were cultured in a humidified atmosphere (95% air, 5% CO 2 ) at 37C in Dulbecco's modified Eagle's medium (DMEM, Biochrome, Berlin, Germany) containing 10 ml of penicillin/streptomycin solution, 1 ml of amphotericin B supplemented with 10% fetal bovine serum (FBS, Biochrome, Berlin, Germany). Upon reaching confluence, the cells were detached using trypsin-EDTA solution. Cells between the passages 7-10 were tested for viability using tryptophan blue and were used for the attachment assay.

Two 24-well plates (Plates A and B) were selected; Plate A was used for the adhesion assay and Plate B was used for the alkaline phosphatase assay.

In Plate A, four specimens of each membrane (n=16) were trimmed to an approximate size of 5x5 mm, placed on the floor of 24-well micro culture dishes and immersed in a serum-free cell culture medium for 1 hour. Thereafter, this medium was replaced by medium supplemented with serum. Using a micropipette, 5 10 4 cells/ml human osteoblast-like cells (SaOS-2) suspended in DMEM supplemented with 10% FBS were seeded on the surface of the barrier membrane. In the case of the bilayered BG membrane, SaOS-2 osteoblasts were cultured on the porous surface.

A similar procedure was repeated in Plate B. Both the plates were incubated for 24 hrs at 37C in a humidified atmosphere of 95% air and 5% CO 2. The entire procedure was done under aseptic conditions in a laminar hood.

MTT (3- [4, 5-Dimethylthiazolyl-2]-2, 5-Diphenyltetrazolium Bromide) Assay

The adhesion assay was carried out in accordance with what was proposed by Mosmann. [18] Briefly, the membranes were retrieved from the wells after 24 hrs, 100 l of 0.5% MTT was added and then incubated at 37C for 4 hours. After incubation, the MTT containing medium was removed from the plate and 100 l of solubilising solution consisting of 20% Sodium lauryl sulfate in 50% dimethylformamide was added to each well to dissolve the formazan crystals formed from the tetrazolium salts. The optical density (OD) of the colored complex formed was read at a wavelength of 650 nm using a spectrophotometer (Molecular Devices, Sunnyvale, USA.). The number of viable cells adhering to the membranes (directly proportional to OD) was calculated based on the readings obtained from the spectrophotometer. The experiments were repeated three times with tetrad samples.

Alkaline Phosphatase Assay (Plate B)

After an initial estimation of cellular protein content, a cellular alkaline phosphatase (ALP) assay was done according to the previously established protocol [19] at 24 hours, 72 hours, and 1 week.

Estimation of Protein

The protein concentration was estimated using the method given by Lowry, et al. [20] using crystalline bovine serum albumin (BSA) as the standard. Briefly, 0.1 ml of cell lysate sample was taken in a clean glass tube. The volume in each tube was filled to 1 ml with water. Then, 5 ml of alkaline copper reagent was added, mixed, and allowed to stand for 10 min at room temperature. A total of 0.5 ml of 1N Folin-ciocalteau phenol reagent was added to each tube, shaken well, and incubated for 20 min at room temperature. The intensity of the blue color developed and was read at 640 nm against reagent blank containing all the reagents except the cell lysate. A set of standards were run in each batch of assays. The amount of protein present in the cell lysate samples was calculated by referring to the standard graph obtained or by plotting the standard graph. A set of standards containing 25, 50, 75, 100, 125, and 150 g of bovine serum albumin were taken in a series of test tubes and treated similarly to that of the sample tubes. The standard graph was drawn by plotting the concentration of the standard protein solution on the x-axis and the optical density on the y-axis.

The protein content in the bone cell lysate was calculated using the formula;



A total of 0.5 ml of p-nitrophenol phosphate substrate and 0.5 ml glycine buffer (pH 10.5) were added into clean glass tubes marked 'blank' and 'test'. The tubes were placed in a water bath at 37C for 5 min. The reaction was initiated by the addition of bone cell lysate to the 'test' tubes and water to the 'blank' tubes and the time was noted. After 30 minutes of incubation at 37C, the reaction was arrested by the addition of 10 ml of 0.02 N NaOH. The tubes were mixed well and the color developed was read at 410 nm against the reagent blank. The alkaline phosphatase activity was expressed as moles of p-nitrophenol formed per hour per milligram of protein.

Addition of EMD

SaOS-2 cells were simultaneously cultured in two other culture plates; membranes were trimmed and placed in 16 wells as described previously. A total of 50 g/ml of EMD dissolved in 10 mM was added to 20 wells of the 24-well culture plate so as to obtain 6 groups: BG + EMD; BM + EMD; HG + EMD; CP + EMD; EMD alone, and Control (SaOS-2 cells in DMEM and FBS).The experimental protocol outlined above was repeated for the cell adhesion (MTT) and ALP assay.

Statistical Analyses

The results obtained were analysed statistically and comparisons were made within each group using one-way analysis of variance (ANOVA) followed by a Bonferroni test. A value of P<0.05 was considered as the level of significance.


   Results Top


Osteoblastic Adhesion on the Barrier Membranes (MTT Assay)

During the experimental period, there was no evidence of toxic residues from any one of the membranes and no signs of bacterial or fungal contamination on the well chamber. Data was represented as the mean standard deviation. The results of the adhesion assay with and without the addition of EMD are summarized in [Table 1] and [Figure 1].

Among the four barrier membranes tested, BG (48.431.32) had the highest osteoblast adhesion at a 24-hour time period, which was significantly higher (P<0.05) when compared with all other barrier membranes. Although, HG (37.646.49) showed increased osteoblastic adhesion when compared with BM (32.64 3.89), the difference was not statistically significant (P>0.05). The osteoblastic adhesion to all collagen membranes (BG, HG, and BM) was significantly higher (P<0.05) when compared with CP (21.802.65).

Following the addition of EMD, there was a slight but statistically insignificant decrease in osteoblast adhesion to the collagen barriers. Osteoblast adhesion to cytoplast was virtually unaffected by the addition of EMD.

At 72 hours and 1 week, no further changes occurred in the adhesion assay.

Alkaline Phosphatase Activity

The results of the ALP activity of the osteoblasts adhering to the membranes, with and without the addition of EMD, at the end of 24 hours, 72 hours, and 1 week are summarized in [Table 2] and [Figure 2].

24-Hour Results

Among the four barrier membranes tested, BG (38.581.69) had the highest alkaline phosphatase activity at 24 hours, which was significantly higher (P<0.05) when compared with all other barrier membranes. Although, HG (33.751.46) showed increased alkaline phosphatase activity when compared with BM (31.251.80), the difference between BM and HG was not statistically significant (P>0.05). Osteoblast differentiation as measured by ALP activity was significantly higher (P<0.05) in all collagen membranes (BG, HG, and BM) when compared with CP (25.66 1.66).

Osteoblasts exposed to EMD alone showed significantly greater ALP activity when compared with the control at 24 hours. Among the various barrier membranes, BG+EMD (48.751.47) showed higher but statistically insignificant alkaline phosphatase activity when compared with the control (44.202.25). HG+EMD (39.404.84) and BM+EMD (37.353.64) showed a lower but not statistically significant ALP activity when compared with the control (44.202.25). There was no statistically significant difference in the ALP activity between the collagen membrane groups. In all these membrane groups, however, ALP activity was significantly higher following the addition of EMD, when compared with the membrane alone.

72-Hour Results

A similar trend of results was observed except that BG+EMD showed a statistically significant increase in ALP activity when compared with the control. The ALP activity was, however, uniformly higher in all the groups when compared with the 24 hr results.

1-Week Results

ALP activity was significantly higher (P<0.05) in BG+EMD (64.783.04) when compared with the control (46.29 1.39) and to all other barrier membranes HG+EMD (55.403.89) and BM+EMD (54.75 4.17). HG+EMD (55.403.89) and BM+EMD (54.75 4.17) showed significantly higher (P<0.05) ALP activity when compared with the control (46.29 1.39).

ALP activity in the CP+EMD (37.46 3.54) group was significantly less (P<0.05) when compared with the control (46.29 1.39) even after a period of 1 week.

At all stages, ALP activity with EMD alone was significantly higher than that obtained when osteoblasts were exposed to an EMD/barrier membranes combination. An EMD/membrane combination showed a significantly higher ALP activity when compared with the membrane alone at every stage. Similarly, EMD/collagen showed a significantly higher activity when compared with EMD/CP.


   Discussion Top


Regenerative periodontal therapy has become a fairly predictable procedure today. Regenerative outcomes are, however, dependent on the recruitment of the progenitor cell population residing in the remaining periodontium in the wound space. Ideally, a barrier membrane should facilitate this process by providing a substrate that can act as a matrix and/or provide signals necessary for the regenerating cells to proceed with the wound-healing cascade of proliferation, differentiation, and tissue maturation. [7],[21] This process begins with cell adhesion, a four step sequence that includes adsorption of glycoprotein to the substrate surface, cell contact, attachment, and spreading. [21] A MTT assay was used in our study in preference to H and E staining as the number of viable osteoblasts adhering to the membrane could be assessed accurately based on the degree of absorbance registered on the spectrophotometer. [18] The initial adhesion of osteoblasts was observed after 24 hrs, as previous studies have established that it is at this time interval that mesenchymal cells exhibit the greatest adhesion to membranes. [8]

The results of osteoblast adhesion to barrier membranes are in agreement with previous studies. [22] Adhesion to collagen barriers was significantly higher as Type I collagen (HG and BM) barrier membranes are made of Type I collagen, while BG has Type I and III collagen, which is a natural substrate for osteoblasts, unlike the biologically inert polymer (CP). SaOs2 cell adhesion to collagen was inversely proportional to the degree of cross links present in the collagen membranes in accordance with previous reports. [23] Thus, the adhesion was greatest with the non cross-linked BG followed by the weakly cross-linked HG and then the glutaraldehyde cross-linked BM. The central role played by collagen in regulating osteoblast differentiation and their alkaline phosphatase activity has been previously established. [24],[25] Thus, the finding that the membrane group showed greater adhesion to osteoblasts and demonstrated greater ALP activity was along expected lines.

The addition of EMD appeared to have no influence on osteoblast adhesion to CP, while marginally reducing the adhesion to collagen barriers. Differential expression of adhesion molecules in osteoblasts could have resulted in this statistically insignificant reduction, although this could not be ascertained in this study. For example, EMD is known to upregulate the expression of only the αvβ3 integrin (not involved in cell adhesion) in the mesenchymal cells. [26]

The initial response of the osteoblasts to the EMD/collagen membrane combination seems to be to mediate adhesion to collagen, thereby resulting in a lag phase during which ALP activity was less than even the control. The significant increase in ALP activity observed in the EMD/collagen groups after the initial lag phase could be explained by the upregulation of Runx2 - cbfa1, which is the master switch that regulates osteoblast differentiation and has been reported in an osteoblast cell line. [27]

To summarise, the EMD/collagen membrane combination modulates osteoblast behavior so that EMD marginally decreases osteoblast adhesion to collagen while collagen downregulates EMD's influence on ALP activity. Together, however, they positively influence osteoblast differentiation, as the ALP activity of all the three EMD/collagen groups was significantly greater than that of the control after 1 week.

The polymer barrier (CP) has been shown to exhibit a minimal biological effect on mesenchymal cells, as it is neither a natural substrate nor a regulator of signaling mechanisms that affect their differentiation. [28] Consequently, osteoblasts adhered poorly to the membrane and the EMD/CP membrane combination did not exhibit significant ALP activity. As the ALP activity of the EMD/CP group continued to be less than that of the control even after 1 week, the biological rationale of using this combination may be questionable.


   Conclusion Top


Osteoblasts adhere strongly to collagen barriers but not to polymer barriers and the adhesion was not affected by the addition of EMD. ALP activity of osteoblasts was significantly increased following the addition of EMD.

One must use caution in interpreting results obtained using an in vitro experimental model, as it cannot recreate the complex interactions of cells that occur in the periodontal wound space. However, there is a necessity to ascertain the biological effects of a combination of regenerative materials before putting them to clinical use.


   Acknowledgments Top


The authors are grateful to Mr. R.C. Vignesh and Dr. S. Sitta Djody of the Department of Endocrinology, Dr. A.L.M Post Graduate Institute of Basic Medical Sciences for their valuable suggestions and guidance. We thank Dr. R. Ravanan Ph.D., Professor, Dept. of Statistics, Presidency College, Chennai for his competent help in statistical evaluation.

 
   References Top

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4.Black BS, Gher ME, Sandifer JB, Fucini SE, Richardson AC. Comparative study of collagen and expanded polytetrafluoroethylene membranes in the treatment of human class II furcation defects. J Periodontol 1994;65:598-604.  Back to cited text no. 4    
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13.Schwartz Z, Carnes DL Jr, Pulliam R, Lohmann CH, Sylvia VL, Liu Y, et al . Porcine fetal enamel matrix derivative stimulates proliferation but not differentiation of pre-osteoblastic 2T9 cells, inhibits proliferation and stimulates differentiation of osteoblast-like MG63 cells, and increases proliferation and differentiation of normal human osteoblast NHOst cells. J Periodontol 2000;71:1287-96.   Back to cited text no. 13    
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23.Baslι MF, Grizon F, Pascaretti C, Lesourd M, Chappard D. Shape and orientation of osteoblast-like cells (Saos-2) are influenced by collagen fibers in xenogenic bone biomaterial. J Biomed Mater Res 1998;40:350-7.  Back to cited text no. 23    
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25.Lynch MP, Stein JL, Stein GS, Lian JB. The influence of type I collagen on the development and maintenance of the osteoblast phenotype in primary and passaged rat calvarial osteoblasts: modification of expression of genes supporting cell growth, adhesion, and extracellular matrix mineralization. Exp Cell Res 1995;216:35-45.  Back to cited text no. 25    
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Correspondence Address:
Avaneendra Talwar
Department of Periodontics and Implantology, Ragas Dental College and Hospital, Chennai
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.49048

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