Versatility of Pleuripotent Undifferentiated Stem Cells Aspirated from Bone Marrow and its Applications in Oral and Maxillofacial Surgery

Abstract

Introduction

Bone marrow (BM) derived pleuripotent undifferentiated stem cells represent a promising population for supporting new concepts in cellular therapy.

Aim

The aim of this study is to evaluate the versatility of pleuripotent undifferentiated stem cells derived from BM aspiration and its applications in oral and maxillofacial surgical procedures.

Materials and Methods

A total of 30 patients out of which 15 were with hard tissue defects (cystic lesions n = 6, post surgical alveolar defects n = 4, peri implant defects n = 3, alveolar clefts n = 2) and 15 soft tissue lesions (leukoplakia and lichen planus n = 6, oral submucous fibrosis n = 7, post traumatic soft tissue loss n = 2) were included in the study on randomized clinical basis. The patients received autologous BM derived mononuclear cells which were being locally delivered into the lesion and followed up. The parameters used were (1) To compare and evaluate the bone regeneration by radiographic assessment at the end of 3rd and 6th month postoperatively. (2) Duration of the procedure. (3) Clinical improvement in the management of soft tissue lesions. (4) Assessment of wound healing by Vancouver burn scar assessment of wound. (5) Safety, postoperative infections and complications.

Results

For hard tissue lesions CT scans and OPG revealed adequate regenerated bone, bridging the defect after 3 months. Hounsfield units of regenerated bone after 6 months were more or less similar to native bone which was statistically significant (unpaired t test = p < 0.05). For soft tissue lesions (1) 7 cases of OSMF showed adequate clinical mouth opening (one way anova test = p < 0.05), reduction in burning sensation and blanching of mucosa, (2) 6 cases of leukoplakia and lichen planus and 2 cases of post traumatic soft tissue defects showed good clinical improvement by Vancouver burn scar assessment of wound index.

Conclusion

The study shows that there is a definite beneficial effect in bone regeneration and soft tissue wound healing with the use of BM-derived mononuclear cells.

Electronic supplementary material

The online version of this article (doi:10.1007/s12663-015-0793-2) contains supplementary material, which is available to authorized users.

Introduction

Stem cell therapy undoubtedly holds much promise for the treatment of many diseases. There is considerable evidence that the human bone marrow supplies the body with not only hematopoietic but also a significant number of mesenchymal stem cells (MSCs) [1]. Bone marrow aspirate has been investigated as a source of mesenchymal stem cells for regeneration of cartilage and other tissues of the musculoskeletal system. Recently, tissue engineering and cell based therapy have provided clinicians with alternative ways of harvesting autologous bone marrow concentrates to treat local bone defects [2].

However, there are currently very few clinical publications/trials reporting the use of pleuripotent undifferentiated stem cells derived from bone marrow harvest for treating hard and soft tissue lesions. There is a wide discrepancy in the literature when it comes to the number of publications dealing with basic science research versus clinical research involving bone-marrow mononuclear cell (BMMNC) or mesenchymal stem cell (BM-MSC). Based on a Medline database review in September 2014 (http://www.pubmed.de), we found 547 articles by using the key words “mesenchymal”, “stem cell”, and “mononuclear cells”, however, when the word “patient” was added, only 159 papers were found; when the word oral and maxillofacial surgery was added only two articles were found. Furthermore, the phrases, “bone marrow”, “concentrate”, and “osteoblast” resulted only in two articles, both of which were in vitro studies. Although using the terms “mesenchymal”, “stem cell”, “bone”, “healing”, and “patient” presented with 11 hits in pubmed, only one of these articles (case report) reported about successful bone healing via MSC derived from the periosteum in an atrophic femoral non-union. All of the other studies were either in vitro, review of the literature, or research dealing with nonosseous tissue defects such as diabetic ulcer and skin defects. In bone marrow aspirates, mesenchymal stem cells represent a very small fraction of the total population of nucleated cells. Studies suggest that 0.001–0.01 % of mononuclear cells, after density gradient centrifugation of the bone marrow aspirate to remove red blood cells, granulocytes, immature myeloid precursors, and platelets, are mesenchymal stem cells [3]. The number of mesenchymal stem cells can be increased through culture expansion, but then the technique is no longer a simple, one-step method of tissue regeneration [4].

From a surgeon’s perspective, there are several advantages to a one step clinical application of bone marrow concentrate [5]. An immediate autologous transplantation of bone marrow concentrate can prevent complications related to the reduced quality of the transplanted cells such as pre-aging (telomere shortening which eventually prompts them to stop dividing and die), reduced viability, or dedifferentiation/reprogramming that is associated with in vitro-cultivation [6].

In addition, the risk for infection is reduced by decreasing the time for cell culture and contamination. Our study reports a case series on the clinical outcomes after autologous mononuclear cells isolated from bone marrow in treating hard and soft tissue lesions.

Patients and Methods

A prospective randomized clinical study was conducted and the protocol was approved by the institutional ethical committee.

Patients Selection

The patients referred to the Department of Oral and Maxillofacial Surgery of the Kamineni Institute of Dental Sciences, Nalgonda with hard and soft tissue lesions in oral and maxillofacial region were selected. The inclusion criteria were: (a) Patients above the age of 12 years. (b) Patients with soft tissue lesions of oral cavity (lichen planus & leukoplakia). (c) Cases with oral submucous fibrosis (OSMF) with Grade III, Grade II and Grade I mouth opening [7] were included in the study and evaluated postoperatively at 1st and 3rd months. (d) Patients with deficient bone (cleft alveolus, periapical bone defects, atrophic mandible, after excision of tumors and enucleation of cysts). (e) Patients with delayed union. Criteria for exclusion of cases were: (a) Patients below 12 years of age. (b) Patients of uncontrolled systemic diseases (haematological diseases), ongoing chemotherapy or radiation therapy. (c) Immunocompromised or immune suppressed patients. A total of 30 patients were selected and divided into two groups A and B. Group A included treatment for hard tissue defects and group B included treatment for soft tissue lesions of oral and maxillofacial region treated from August 2012 to July 2014. Thirty consecutive patients (age 30.8 + 8.921 years, range 19–50 years) were included in the study and randomly bone marrow was harvested from iliac crest and mandible. Moreover, all procedures were conducted in a single surgery.

Method of collection of data: (1) All patients were informed about the risks of the procedure and possible complications. (2) Written and informed consent were taken. (3) Before treatment thorough clinical and radiographic examination was made in patients, using orthopantomograph, cone beam computed tomograms and computed tomography scans. Bone density was evaluated of the regenerated bone and native bone by CT scan in Hounsfield units (HU).

Isolation and Administration of Autologous Mononuclear Bone Marrow Cells

Patients were evaluated for safety i.e., laboratory tests (Hb  %, RBC, WBC, platelets, CT, BT) preoperatively. Bone marrow (mean = 60 ml) was harvested under aseptic conditions from the anterior iliac crest (n = 20) and bone marrow (7 ± 0.95 ml) from mandible (n = 10) using Jamshidi needle. Bone marrow was diluted with phosphate buffered saline and centrifuged at 2200 rpm for 25 min. Bone marrow was than processed in a closed system for volume reduction. Bone marrow mononuclear cells (BM-MNC) layer (buffy coat) was separated, their sterility and viability was maintained before injection. Viability of concentrate was checked by tryptan blue staining. Volume reduced sample was processed to have rich 1–5 ml fraction of MNC (haemopoetic stem cells and mesenchymal stem cells and endothelial progenitor cells). MNC count was counted for each patient. The concentration of mononuclear cells in the bone marrow aspirate and in the separated bone marrow concentrate was analyzed by an automatic cell counter. The 30 patients were delivered with mononuclear cells the same day of harvesting. An aseptic technique of delivery was performed, where the cells were given in a sterile syringe which was injected into the lesion.

Technique

For an anterior approach from iliac crest, manually palpate to locate the anterior superior iliac spine and make a 2-mm incision with a #11 blade. The initial trajectory should be medially in line with the pelvic wing as gauged by the inner and outer tables. The needle should be aimed posterior to the iliac tubercle for entry into the medullary canal just beneath it (Fig. 1a). Gently tap the needle into the cortex of the bone and use a mallet to further the needle in the bone. Either 1 or 3 aspiration holes with needles can be used.

Fig. 1.

a Bone marrow harvest using Jamshidi needle from iliac, b step incision made along external oblique ridge, c bone marrow harvest from mandible

Before the mandible aspiration the inferior alveolar nerve was blocked with 2 % lidocaine with 1:80,000 adrenaline. To facilitate insertion of the aspiration needle into the mandible, a small step incision of 1 cm was made on the oral mucosa along the external oblique ridge of the mandible (Fig. 1b), and a small hole was drilled into the marrow portion with the use of a small round bur. After confirming that blood was flowing out of the hole, an 18-G marrow aspiration needle was inserted into the marrow cavity of the ascending ramal area of the mandible (Fig. 1c).

Radiological Evaluation

All cases in group A were subjected to CT scan preoperatively and 3rd month postoperatively (Fig. 2a, b). The CT examinations were performed using a Siemens Somatom AG CT scanner (Siemens, Erlangen, Germany) at a slice thickness of 0.6 mm. We analyzed DICOM data of the tomogram using the computer Software Version Syngo CT 2006A. The CT examination and evaluation involved both morphological analysis and bone density analysis in HU. The CT value of compact bone was taken in native bone and regenerated bone in HU. In orthopantomograph, the cases in which the bone opacity was clearly far lower than that of the native bone were marked (−), cases in which the bone opacity was close to that of the native bone but still lower were marked (+), and cases in which the bone opacity was equal or nearly equal to that of the native bone were marked (++).

Fig. 2.

a Preoperative Dentascans of ridge augmentation, b 3 months postoperative Dentascans of ridge augmentation

Results

Total of 30 patients were included in the study out of which 20 were male and ten were female patients. Overall the age group of patients ranged from youngest 19 years to oldest 50 years of age, with the mean age of the patient being 30 years and mean ± standard deviation = 30 ± 8.921. Bone marrow was harvested from iliac bone and mandible, further cell processing was carried out. Twenty samples were harvested from iliac bone and 10 samples were harvested from mandible based upon randomized basis.

Mean of bone marrow harvested from iliac bone = 60 ml. Mean of bone marrow harvested from mandible = 7 ml. Mean of bone marrow concentrate obtained from iliac bone = 5.35 ml. Mean of bone marrow concentrate obtained from mandible = 1.0 ml. Mean of mononuclear cells from iliac bone = 2.56 × 10⁸. Mean of mononuclear cells from mandible = 1.74 × 10⁷ (Table 1).

Table 1.

Mean comparison of concentration and viable cell percentage (%) from iliac crest and mandible

	Vol. (mean)	No. of MNCs	% viability of cells
Iliac crest	60 ml	2.56 × 108	97.35
Mandible	7.0 ml	1.74 × 107	96.9

Statistical Analysis

For the parameters new bone (new bone formation) values were expressed in HU of the native and regenerated bone (Table 2). For statistical analysis, an unpaired t test for paired group data was used. Fifteen patients of group A were included for analysis. A p < 0.05 is considered as a significant result.

Table 2.

Evaluation of bone opacity on OPG and Hounsfield units by CT examination at 3rd and 6th month postoperatively in group A

S. no.	OPG 3rd month		OPG 6th month		Hounsfield units
	Double positive	Positive	Double positive	Positive	Mean of native bone	Mean of regenerated bone
1	✓		✓		1256	1098
2	✓		✓		1367	1034
5	✓		✓		1114	998
6		✓	✓		1456	1056
7	✓		✓		1343	1114
8	✓		✓		1267	1078
11	✓		✓		1221	1081
14	✓		✓		1108	892
16		✓	✓		1250	980
21	✓		✓		1214	1096
23	✓		✓		1198	1054
27	✓		✓		1290	1108
28	✓		✓		1100	1032
29	✓		✓		1189	989
30	✓		✓		1097	838

Mean of bone marrow harvest from iliac bone = 22.25 min. Mean of bone marrow harvest from mandible = 33.5 min (Graph 1 of supplementary material).

Cases with OSMF classification of group III, group II and group I mouth opening were included in the study and evaluated postoperatively at 1st and 3rd months (Table 3).

Table 3.

Assessment of mouth opening in OSMF patients

S. no.	Preop	1 month postop	3 months postop	Difference in mouth opening
1	22	23	27	5
2	26	26	30	4
3	27	31	31	4
4	22	23	27	5
5	26	26	31	5
6	28	28	33	5
7	31	32	35	4

Wound quality was evaluated by the Vancouver Burn Scar Assessment Scale (VBSAS) [8], which rates the quality of a wound in terms of pigmentation, vascularity, pliability, and height/depression at 1st week postoperatively, 1st month postoperatively and at 3rd month postoperatively (Table 4).

Table 4.

Vancouver burn scar assessment of wound at Time of (a) 1 week (b) 1 month (c) 3 months

S. no.	Pigmentation	Vascularity	Pliability	Contracture	Height
(a)
1	Hyper	Purple	Firm	Absent	Flat-nl
2	Hyper	Red	Firm	Absent	Flat-nl
3	Hyper	Purple	Firm	Absent	Flat-nl
4	Hyper	Purple	Firm	Absent	Flat-nl
5	Hyper	Red	Firm	Absent	<2 mm
6	Hyper	Red	Firm	Absent	Flat-nl
7	Normal	Normal	Firm	Absent	Flat-nl
8	Hyper	Red	Firm	Absent	Flat-nl
9	Hyper	Normal	Firm	Absent	Flat-nl
10	Hyper	Normal	Firm	Absent	Flat-nl
11	Normal	Red	Firm	Absent	Flat-nl
12	Hyper	Purple	Firm	Absent	Flat-nl
13	Hyper	Red	Normal	Absent	Flat-nl
14	Hyper	Purple	Normal	Absent	Flat-nl
15	Hyper	Purple	Normal	Absent	Flat-nl
(b)
1	Normal	Red	Suppleness	Absent	Flat-nl
2	Normal	Normal	Suppleness	Absent	Flat-nl
3	Normal	Red	Normal	Absent	Flat-nl
4	Normal	Normal	Normal	Absent	Flat-nl
5	Hypo	Pink	Normal	Present	<2 mm
6	Hypo	Pink	Yielding	Present	Flat-nl
7	Normal	Normal	Suppleness	Absent	Flat-nl
8	Normal	Normal	Suppleness	Absent	Flat-nl
9	Normal	Normal	Suppleness	Absent	Flat-nl
10	Normal	Normal	Firm	Absent	Flat-nl
11	Normal	Red	Normal	Absent	Flat-nl
12	Normal	Normal	Firm	Absent	Flat-nl
13	Normal	Pink	Normal	Absent	Flat-nl
14	Normal	Normal	Normal	Absent	Flat-nl
15	Normal	Pink	Normal	Absent	Flat-nl
(c)
1	Normal	Red	Suppleness	Absent	Flat-nl
2	Normal	Normal	Suppleness	Absent	Flat-nl
3	Normal	Red	Normal	Absent	Flat-nl
4	Normal	Normal	Normal	Absent	Flat-nl
5	Hypo	Pink	Normal	Present	<2 mm
6	Hypo	Pink	Yielding	Present	Flat-nl
7	Normal	Normal	Suppleness	Absent	Flat-nl
8	Normal	Normal	Suppleness	Absent	Flat-nl
9	Normal	Normal	Suppleness	Absent	Flat-nl
10	Normal	Normal	Firm	Absent	Flat-nl
11	Normal	Red	Normal	Absent	Flat-nl
12	Normal	Normal	Firm	Absent	Flat-nl
13	Normal	Pink	Normal	Absent	Flat-nl
14	Normal	Normal	Normal	Absent	Flat-nl
15	Normal	Pink	Normal	Absent	Flat-nl

Discussion

The use of Mesenchymal stem cell (MSC) without an expansion step relies on the separation of sufficient numbers of MSC and concentration of the MSC fraction in a reduced volume that can be injected [9]. Since the MSC cannot easily be identified within the Nuclear Cell (NC) fraction without additional laboratory analysis, the complete NC fraction has to be used, i.e. with all white blood cells. There is no evidence whether the injection of a mixed cell population of nuclear cells influences performance. One could postulate that the disintegration of white blood cells might cause an inflammation reaction with negative side effects [10]. On the other hand, cell signalling and interaction with other progenitor cells within the NC fraction might be beneficial [11]. Consequently, a mixture of mononuclear cells, lymphocytes and polynuclear leukocytes might support the growth of CFU-F [12].

Sauerbier et al. [13] found 17.2 × 10³ + 13.5 × 10³ white blood cells (WBC)/µl. The BMAC process resulted in 79.4 × 10³ ± 45.8 × 10³ WBC/µl with 41.4 ± 15.6 colony forming units/1 × 10⁶ mononuclear cells. Our study has similar findings with other published trials (orthopaedic and cardiac) having no adverse reactions, mortality or any other risk factors involved with MNCs administration of up to 150–200 million cells [14].

In group A, radiographically on a panarogram the bone opacity was close or some times more than that of the native bone. The opacity in the lesion was evident following 3 months and progressively increased to the maximum by 6 months. Clinical and radiographic analyses of treatment sites demonstrated that the cell therapy accelerated the regenerative response as determined clinically, radiographically (Fig. 3a, b). The mean CT value of compact bone was taken in native bone group and regenerated bone in HU. The difference in proportions of qualitative parameters are compared using unpaired ‘t’ test. The findings are somewhat significant (p < 0.05). The CT Hounsfields value of MSC therapy treated bone was more or less close to that of native bone in bone defects as in group A.

Fig. 3.

a Preoperative cystic lesion, b 3 months postoperative enucleation and cellular therapy

The operating time was measured from the time of bone marrow harvesting to the end of procedure. The cellular processing for isolation of mononuclear cells was considered and operative procedure was included. Vacuum aspiration of autologous bone marrow was 15–30 min. Isolation of Bone marrow concentrate by centrifugation takes about 20 min. Cell viability/MNC count takes about 10 min. There is no prolonged operation time because the bone marrow concentration process runs parallel to the surgery by an additional trained person. Considering this, the surgeon has to only aspirate the bone marrow and inject the stem cell concentrate in the hard and soft tissue lesion which is much faster than performing an autologous bone grafting or excision to reduce operating time.

In oral submucous fibrosis, intralesional stem cell therapy has shown significant improvement in mouth opening (Fig. 4a, b) and gradual decrease in burning sensation and blanching of mucosa. Stem cell therapy is primarily aimed at neoangiogenesis by releasing cytokines and growth factors (paracrine effect). This may result in increased free radical scavenging by antioxidants (either naturally occurring or extraneous); neoangiogenesis may also facilitate the removal of senescent cells from the lesions by supplying more number of scavenging defense cells, and reversal of hypoxia in the diseased tissue.

Fig. 4.

a Preoperative mouth opening, b 3 months after cellular therapy

Stem cell therapy may help to stimulate resident tissue stem cells to transform into new fibroblasts, which may help in removal of disintegrated biochemically and morphologically altered collagen fibers [15]. Sankaranarayan et al. [15] injected 175 million BMMNCs into the area affected. The paramaters such as blanching, fibrous band which improved, 4 weeks after the injection and a significant improvement in mouth opening correlates with our findings in which we infiltrated BMMNCs. An improvement in mouth opening of 5 mm and reduction in clinical findings such as burning sensation and blanching of mucosa following 3 months were observed. In OSMF there was gradual reduction in blanching, improved/better suppleness of the mucosa, decrease in the burning sensation while consuming spicy food, increase in the mouth opening. The above mentioned results were found to be sustained in the follow-up period. Assessment of clinical findings in the immediate postoperative period was done. The patients were discharged the following day itself. The patients were reviewed every week for the first 4 weeks and then every month for the next 6 months. Cases started showing improvement from the fourth week onward. In premalignant lesions the patients started showing good improvement in clinical signs and symptoms from 1 month posttreatment followed by cellular therapy.

In our study the mononuclear cells (MNCs) were easy to procure from the bone marrow and had an average viability of 99 % before initial transplantation which is similar to what is described in previous trials. Their viability was evaluated by tryptan blue staining. Trypan blue molecule is cell membrane impermeable and therefore only enters cells with compromised membranes. Upon entry into the cell, trypan blue binds to intracellular proteins thereby rendering the cells a bluish color. The trypan blue exclusion assay allows for a direct identification and enumeration of live (unstained) and dead (blue) cells in a given population.

Our patients with this cellular therapy did not report any complications of bone marrow harvesting from iliac crest and mandible. The patients were readily mobilized after the bone marrow harvest immediately next postoperative day. Thus the safety, feasibility and tolerance of autologous MNCs transplantation in patients were established. Specific complications from the method of MNSC therapy did not occur in any of the 30 patients. In particular, no complications were observed concerning excessive new bone formation, infections, tumor induction or morbidity. The treatment of bone healing disorders with purified bone marrow is not a new therapy. Attempts at direct bone marrow inoculation fail due to the volumes necessary for successful therapy. For this reason, the concentration of mononuclear cells with the help of density gradient centrifugation was developed. With this procedure a mixed population of different cells is produced including mesenchymal precursor cells: colony forming units-fibroblastic (CFU-F) and hematogenic stem cells (CD34 cells) [3, 9].

In addition to these first positive results, the present study shows that no significant specific risks and complications are connected with the intra-operative removal and purification of progenitor cells in a relevant number of patients. The weakness of the present study is that it has short follow-up period. Accordingly, long-term post-surgical examinations and studies are necessary on statistically relevant patient numbers. The good results in premalignant lesions, OSMF, periapical cystic lesions which were achieved with pleuripotent stem cells purified in the laboratory makes this simple procedure a valuable addition to the previous therapy options.

There is no sign of any systemic or local toxicity related to the treatment, such as systemic fever, anemia, local calcification, or scleroma in the injection area developed in any of these patients who underwent local stem cell delivery. Safety measures were taken during this study by informing the patients about all details of the study and the patients signed an informed consent that were taken from all patients before participating in the study.

Conclusion

The study shows that there is a definite beneficial effect in bone regeneration and soft tissue wound healing with the use of BM-derived mononuclear cells. A non-randomized clinical study, quantification of cells dose were few limitations, nevertheless our results showed a significant improvement in clinical and radiological findings. Our results demonstrated that there is a rationale for a clinical application of bone marrow derived mononuclear cells in the treatment of osseous defects. The intraoperative harvest procedure is a safe method and does not significantly prolong the time of surgery. There were no specific complications within the short follow-up period and a simple intra-operative use of the system for different forms, the on-site preparation of the bone marrow cells within the operating theater eliminates the specific risk of ex vivo cell proliferation and has a safety advantage in the use of autologous cell therapy for bone regeneration and wound healing. Additional studies should be completed to determine efficacy and long term follow up.