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Evaluation of the effect of adipose tissue-derived stem cells on the quality of bone healing around implants.

Golab KG1, Kashani IR2, Azami-Tameh A3, Zaminy A4, Nik IN5, Nik SN6.
  • 1Private practice , Tissue engineering , Tehran , Iran.
  • 2Department of Anatomy , School of Medicine, Medical Sciences, University of Tehran , Tehran , Iran.
  • 3Anatomical Sciences Research Center , Kashan University of Medical Sciences , Kashan , Iran.
  • 4Department of Anatomy , School of Medicine, Guilan University of Medical Sciences , Rasht , Iran.
  • 5Faculty of Life Sciences , University of Manchester , Manchester , United Kingdom.
  • 6Private practice, Implantology , Manchester , United Kingdom.

 

ABSTRACT

Purpose/Aim: This study evaluates the efficacy of grafted adipose-derived stem cells (ADSCs) on blade-type implants in improving osseointegration in rat femurs using a low-density bone model. Materials and Methods: After isolating and expanding ADSCs, twice-passaged cells were seeded on blade-type implants on culture plates. Osteogenic induction of grafted cells began after attaching cells to the prepared titanium surfaces and it continued for 4 days. The scaffolds were then implanted in the femurs of Wistar rats. Osteogenic differentiation of these cells was confirmed using polymerase chain reaction (PCR) and alizarin red staining of the mineralized extracellular matrix. After 8 weeks, histological and histomorphometric evaluations of undecalcified resin sections (bone–implant contact [BIC] % and bone mineral index [BMI]) were performed using light microscopy and scanning electron microscopy. Results: Alizarin red staining in conjunction with gene expression results confirmed osteogenic differentiation. Histomorphometric assessment using scanning electron microscopy demonstrated improved BIC% and BMI near the treated surface compared with the untreated surface. Conclusions: The complex of differentiated grafted ADSCs and extracellular matrix and the macrodesign and microdesign of the implant can improve osseointegration in low-density bone.

KEYWORDS: Adipose-derived mesenchymal stem cells; bone; implant; osseointegration; titanium

PMID: 26691556

 

Supplement:

Osseointegration of titanium implants is a necessary prerequisite for their proper function. It depends upon several factors which can determine bone regeneration near the implants. Mesenchymal cells and their differentiation towards osteoblastic lineage are among those factors which play crucial role in the bone healing near titanium surface. Although a myriad of research has been done to improve the titanium surface microdesign to improve healing abilities of bone, it seems that biological additives to the titanium can be a promising field. Several biochemical elements have been incorporated to the titanium surface in order to increase the affinity of mesenchymal cells to these surfaces or enhance the bone regeneration abilities of these cells. The imminent weak point of this technique, is the possible biodegradation of the biological molecules in contact with the tissue environment(1). Several cell lines have been proposed for grafting on implants. Osteoblasts and periosteal stem cells have difficulties in harvesting procedures. Bone marrow stem cells have complicated harvesting approach which may need general anesthesia. As a potential alternative source for regenerative cells we assumed that grafting the adipose derived stem cells on the surface of the titanium surface and their proliferation rate after insertion into the bone,  may  act as a reliable source to provide sufficient osteoblasts(2). Grafting cells seems to be potentially effective, if we consider that in many osteoporotic patients shortage of mesenchymal cells is prevalent evidence.

Based on these concepts we considered to find a rich source of mesenchymel cells to harvest cells from. Donor site should posses high amount of these cells and harvesting of cells should be of minimum invasion. Previous studies had confirmed the osteogenic abilities of mesenchymal cells from adipose tissues. The adipose tissue was harvested from rat and the stem cells were isolated by collagenase type 1. Using centrifugation, we collected stem cells and seeded them in culture plates to isolate the fibroblastic shape stem cells. Culture medium for passaging the stem cells consisted of Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and antibiotics. After two passages we collected these cells from culture plates using trypsin and EDTA. Vitality of these cells confirmed and seeded on specially designed titanium implants. The sandblasted and acid etched implants were designed to be inserted in rat femur with high primary stability. After seeding the cells and their attachment on the titanium surface which took nearly twenty-four hours, the culture medium was replaced with osteogenic medium with the aim of launching the osseodifferentiation of the stem cells (Figure 1, 2). In our study we used dexamethasone, β-glycerol phosphate and ascorbic acid as osteogenic component. Previous studies had shown 4 days of oseteogenic induction as the best condition for osteogenesis (3).

Rat femur with high amount of poor quality bone marrow can be considered as a good model for this study. After primary oseodifferentition of grafted cells, the implants were inserted into the rat femur for two months healing period. The rats were sacrificed after 8 weeks. Undecalcified resin sections were made to measure the bone implant contact and bone mineral index near the implants. Thinned and polished sections were prepared for electronic microscope evaluation using gold plating techniques. Back scattered detector of scanning electron microscope clearly illustrated the bone healing on titanium surfaces (Figure 3) and provided enough fields for measuring the calcium content of the samples. Close contact of osteoblast with titanium confirmed using high magnification (Figure 4). Upon completion of electron microscope evaluations, the specimens were dyed with toluidine blue and analyzed them under conventional light microscopes.

Statistical analysis of our data demonstrated that although there was an improvement in the bone regenerative abilities in the surfaces with grafted cells, there were no significant difference with control surfaces. These results may necessitate further studies with the aim of enhancement in the number and potentials of mesenchymal cells. Furthermore, biochemical techniques which mark grafted cells may be valuable to help trace these cells inside the body (4).

We believe that our technique to improve bone quality around implant does not have the limitations of biodegradation of biochemical elements in the surrounding tissue. This concept can be improved with further studies.

 

References:

  1. Tayalia P, Mooney DJ. Controlled growth factor delivery for tissue engineering. Adv Mater. 2009;21(32-33):3269-85.
  2. Levi B, Longaker MT. Concise review: adipose-derived stromal cells for skeletal regenerative medicine. Stem Cells. 2011;29(4):576-82.
  3. Castano-Izquierdo H, Alvarez-Barreto J, van den Dolder J, Jansen JA, Mikos AG, Sikavitsas VI. Pre-culture period of mesenchymal stem cells in osteogenic media influences their in vivo bone forming potential. Journal of biomedical materials research. 2007;82(1):129-38.
  4. Golab KG, Kashani IR, Azami-Tameh A, Zaminy A, Nik IN, Nik SN. Evaluation of the effect of adipose tissue-derived stem cells on the quality of bone healing around implants. Connective tissue research. 2016;57(1):10-9.