Histomorphometric and Biochemical Evaluations of Stem Cells for Osteogenesis and Neogenesis

Research Article

Austin J Dent. 2015; 2(3): 1024.

Histomorphometric and Biochemical Evaluations of Stem Cells for Osteogenesis and Neogenesis

Alkaisi Amera*

Department of Oral and Maxillofacial Surgery, University of Anbar, Iraq

*Corresponding author: Alkaisi Amera, Department of Oral and Maxillofacial Surgery, University of Anbar, Iraq

Received: July 02, 2015; Accepted: September 01, 2015; Published: September 10, 2015


The purpose of this study was to quantify bone tissue and blood vessel area in the central part of regenerate after transplantation of Stem Cells From Human Exfoliated Deciduous teeth (SHED) in rabbit Mandibular Distraction Osteo Genesis (MDO) in comparison to conventional MDO, As well to measure serum Alkaline Phosphatase (ALP) level and to correlate the 3 parameters. A randomized controlled trial was carried out with 18, 3 - 5.5 month old male New Zealand white rabbits, weighing 2.8 ± 0.2 kg. All animals were divided into 2 groups with 9 animal each (A, as experimental group and B, control). Six million cells were transplanted into the distracted area during the osteotomy period. After a 4-days latency period, a total of 6 mm was distracted for 6 days. The newly formed bone in the central area of the DO regenerates were analyzed histologically, histomorphometrically and biochemically at weeks 2, 4, and 6 postoperatively. All data were analyzed using non parametric Mann-Whitney U test. Spearman correlation test was used to correlate between the 3 variables. The significance was accepted when P is less than 0.05. More bone was observed in the central area of SHED transplanted group histologically and Histomorphologically, the percentage of newly formed bone after 2 weeks were 16 and 0; after 4 weeks, 39 and 22; and after 6 weeks, 27 and 23% in the SHED and control groups respectively. The difference between the groups was statistically significant (P = 0.024). The calculated percentage of blood vessels area at 2 weeks SHED and control group were 5.9 and 4, at 4 weeks 34.42 and 14.64 and at 6 weeks 17.64 and 19% respectively, however; the difference between the groups was statistically not significant P = 0.489. The mean difference of serum alkaline phosphatase level of SHED and control groups were 128.33 and 42.66 respectively with significant result, p< 0.001. A positive correlation was found between the volume density of bone and blood vessels P = 0.014, volume density of bone and ALP, P = 0.029 and no correlation between blood vessel volume and ALP level, p = 0.372.

Conclusion: The results of this study suggest that stem cell therapy accelerate callus and bone formation in mandibular osteodistraction, and increase blood vessels, therefore facilitated consolidation. It may be a valuable approach for clinical osteo distraction enhancement, especially in sites without enough osteogenic potential and vascularization.

Keywords: Distraction osteogenesis; Stem cells; Osteogenesis; Neogenesis


Attention was directed toward mandibular Distraction Osteogenesis (DO), due to expanding demand for minimally invasive orofacial reconstructive techniques. Therefore, an adequate understanding of the biologic events, which take place during DO, is necessary to effectively modulate and manipulate the bony regeneration. The clinical goals of DO research include speedier formation of new bone, shorter fixation time, enhanced bone quality, and minimized risk of nonunion of the osteotomized edges. A multipotent postnatal stem cells have been identified, reside in the pulp of human primary (baby) teeth, these cells have been designated as SHED (Stem Cells from Human Exfoliated Deciduous teeth) [1]. They are isolated from a disposable source (i.e., exfoliated deciduous teeth) noninvasively and retain their potentiality after in vitro expansion, offer a significant advantages. SHED have been found to divide continuously and could be differentiated into a variety of other cell types including nerve, fat, bone, and tooth generating cells. Deciduous teeth may provide an ideal source of stem cells to repair damaged tooth structure, to regenerate bone and perhaps treat nerve damage. The use of SHED might bring advantages for tissue engineering over the use of stem cells from other sources as they have higher proliferation rate [1,2]. The use of multipotent postnatal stem cells from a disposable source that can be readily isolated noninvasively and retain their potentiality after in vitro expansion, offers a significant advantages. Importantly, these cells form a functional vasculature as well as connective tissue secreting cells of the soft and hard tissues of the tooth [3] however there are few studies regarding regeneration of bone defects using MSCs [4,5]. In DO, Kitoh et al. and Jiang et al. [6,7] found more new bone formation and faster calcification in the distracted callus in autologous BMMSCs injected distraction gap of the mandible when compared with the rabbits received saline injection. However very few researches found in the literature using SHED in bony defect and DO, de Mendonca Costa et al. [8] evaluated the capacity of human Dental Pulp Stem Cells (hDPSC), isolated from primary teeth to reconstruct a large-sized cranial bone defects in Non Immune Suppressed (NIS) rats, they found that hDPSC is another cell resource for correcting large cranial defects in rats and constitutes a promising model for reconstruction of human large cranial defects in craniofacial surgery to recruit murine host osteogenic cells [1,8]. Seo et al. [9] found that when SHED is transplanted into a critical-sized calvarial defect area, they generate bony tissue to repair the defect. However, only our previous study, Alkaisi et al. [10] was found using SHED in DO for the same purpose, therefore this study may be the 2nd one using SHED in DO to enhance bone formation. The utilization of DO in the craniofacial skeleton has been steadily increasing for a variety of reconstructive options [11-14] since its original discovery over a century ago by Codivilla [15]. Yet since its initial application for lengthening the mandible in congenital hypoplasia by McCarthy [16] in 1992, we still lack discrete, quantitative metrics to evaluate either the degree of success or failure of the technique. Establishing accurate and reproducible parameters to gauge Regenerate Healing (RG) during Mandibular Distraction Osteogenesis (MDO) should lead to advances in proper application of this technique and improved distraction protocols for enhanced clinical use. Experimental animal studies assessing regenerate healing in MDO using histological analysis have often been subjective and qualitative, or “semi” quantitative [17-19], lacking the measurable precision required to reproducibly determine outcomes. Few animal studies were found using histological measurements in their analysis [10,20, 21]. Several parameters have been used to evaluate regenerate progress include, percentage of new bone volume, cartilage and fibrous tissue, number of blood vessels, number of osteo blasts and osteocytes and others. Ethically it is difficult to apply same method of histomorphometric measurement in human; however there are 2 studies found in the literature measuring some histological parameters in vertical DO using a small biopsy [22,23].The need for non invasive technique to follow and evaluate the stage of regenerate maturation, will help both surgeon and patient to optimize the treatment. Alkaline Phosphatase (ALP), is the main glycosylated protein present in the bone, bound to osteo blast cell surfaces via a phosphoinositol linkage and found free within mineralized matrix [24] as well present in a soluble form in body fluids, including blood [25]. Four isoenzymes exist: intestinal, placental, germ cells and Tissue-Nonspecific, The Tissue-Nonspecific ALP (TNAP) has three isoform, expressed in bone, liver and kidney [26]. It is commonly used as a biochemical marker to assess osteo blast activity and plays as an undefined role in mineralization of bone [24]. Generally speaking, ALP expression increases during early osteogenesis, but when mineralization is advanced, its expression decrease; it is possible to assess Alkaline Phosphatase activity (ALP) expression and stain for matrix mineralization [27]. The purposes of this study were, to quantify bone healing in MDO with transplanted SHED compared to non transplanted one, using a 3, consolidation period [10], to quantitatively measure the bone tissue area in the Region of Interest (ROI), referred as Tissue Area (TA) within the central region of the regenerate after unilateral MDO+SHED in comparison to MDO, to measure blood vessel area and to correlate the amount of newly formed bone with the vessel area. A further specific aim was to measure serum ALP level and to correlate it with our two parameters used aiming to have a new non invasive parameter, can be applied clinically for human DO. We hypothesized that there are significantly higher bone formation, vascularization and serum ALP level in the SHED transplanted group than non transplanted one with significant positive correlation between the variables.

Materials and Methods

Animal care

Ethical approval was obtained from animal ethics committee Universti Sains Malaysia number: USM/Animal Ethics Approval/2010/ (58) (226). This experimental study was carried out with 18, 3 - 5.5 month old male New Zealand white rabbits, weighing 2.8 ± 0.2 kg. All animals were divided into 2 groups, 9 animal each (A as experimental group and B, control). Rabbits were housed in a pathogen-free separated cages with pellet and water.

Surgical procedure

General anesthesia was achieved via intramuscular injection of 35 mg/kg ketamine and xylazine (5 mg/kg). Local administration of 2% lidocaine (Astra, USA) at the surgical site was performed. Right mandible of the rabbits was exposed through a longitudinal incision on the inferior border. A vertical osteotomy was carried out between premolar and mental foramen and a custom-made internal with external activation part distraction device (A SUPER screw palatal expander (Ortho-Care UK Ltd), 12 +12 mm length stainless steel device with full distraction capacity of 12 mm after modification), was fixed to either side of the osteotomy cut and the surgical exposure was primarily closed in layers, the procedure performed according to Alkaisi et al. method [10]. The wound was cleaned with povidon iodine antiseptic and neomycin ointment (antibiotic) was applied.

Distraction osteogenesis protocol

Distraction osteogenesis protocol was 4 days latency period, distraction at a rate of 1 mm/day continued for 6 days resulting in a final lengthening of 6 mm. After the completion of distraction the animals were monitored at 2, 4 and 6 weeks post operatively for consolidation.

Stem cells preparation

SHED were isolated, cultured, characterized and expanded in vitro [10]. About 4-6, T75 tissue culture flasks 80-100% confluent were passaged, cells were counted and adjusted to 6×106. Group A, 6×106 SHED were transplanted in the osteotomy site (at osteotomy period) as an experimental and no transplantation were deposited in group B as a control.

Follow up

The animals were monitored at 2, 4 and 6 weeks period postoperatively. All animals were given an intramuscular injection of Baytril (Enrofloxacin) 10 mg/kg/day (Bayer, Shawnee Mission, KS, USA) once daily and Tramadol hydrochloric (UNICHEM-India), 2mg/kg once daily for pain relief for 7 days.

Histological examination

Each right hemimandible was dissected free of the surrounding tissue immersed in 4% formalin for 24 hours, decalcified in 10% solution of nitric acid for 4 days and automatically processed in a machine for dehydration then embedded in paraffin wax. Sections, 5μm in thickness were cut bucolingually with a microtome and stained with haematoxylin and eosin for light microscopy examination. New bone formation, blood vessels, and fibrous tissue in the central area were assessed.

Histomorphometric examination

All samples were analyzed histomorphometrically using Zeiss image analysis system (Carl Zeiss, MMI 0684-Germany). The images of the histological sections were scanned by the system and saved on a computer. Using computerized image analysis system software, Regions of Interest (ROI), area of 1mm² in the central area of the regenerate was designed, outlined and calculated in square micrometer (Figure 1). New bone trabeculaes outlined, measured and their percentage was calculated. Blood vessels were outlined, measured and the percentage of the vessel area was calculated (Figure 2). Vascular histomorphometry was performed according to Amir et al. [23] as follow: Pictures of the Region of Interest (ROI), were taken in which soft connective tissue and blood vessels were identified. The vascularization was quantified as follow: A blood vessel was defined as a lumen of various sizes, lined by elongated endothelial cells, sometimes surrounded by flattened or cuboidal smooth muscle cells. Occasionally, identification was facilitated by the presence of red blood cells in the lumen (Figure 3). Determination of the volume density of blood vessels defined as the percentage of total soft connective tissue area taken in by blood vessels. Blood vessel volume was calculated as [total blood vessel area/total soft tissue area] x 100%. The measurements were performed at 10 and 20xmagnification by two investigators (A.A and N.A) who were unaware of the origin of the sections. Of each biopsy, 3 sections at different depth were measured and the values averaged.