There were no surgical complications at Stem Cell Group and all patients presented good scar aspect and little inflammatory process at the postoperative follow-up. None of them presented dehiscence (Figs. 5–7).
Statistically there was an association of the lip classification between the groups. The SCG presented higher percentage of “negative” classification (−) in soft tissue inflammatory process (P=0.001), transitory hypertrophy scar (P < 0.001), fibrosis between hard and soft palate (P < 0.001).
According to the palate evaluation there was no statistical difference between the groups (P = 0.471).
The group with stem cells injection presented less postoperative complications and fibrosis.
Facial tomography was done in the first patient to evaluate the osseous development. It was done 2 years of postoperative. The tomography result demonstrated a maxillary alignment, the alveolar cleft became smaller. However, it was no evidence of osseous development.
The lip and palate cleft treatment is always a challenge in multidisciplinary specialties to reach better results. The surgical protocol applied in the patients of Stem Cell Group and Control Group was similar and followed the conventional technique of the professional group.10
Nowadays, other technologies as bioengineering and tissue engineering are growing and been developed in medical procedures.7,9,11
Experimental studies with the use of stem cells already demonstrated important results of regenerative, neovascular, anti-inflammatory and tissue neoformation.4,5,8,12
This study had the purpose to demonstrate the advantages of stem cell into tissue healing at patients with lip and palate cleft. The surgical protocol was used in all lip and palate cleft patients studied, part of them had stem cells injection and part had only the conventional treatment done, considered as control group.10
It was imagined the possibility of increasing the results quality acting in the inflammatory process, muscular function, osteogenesis process, and scar healing after the injection of stem cells. No clinical data (for cleft lip and palate) were found in the literature.
In two experimental studies with rabbits to reconstruct craniofacial bone at where stem cells were used, from adipose tissue1 and bone,12 the results demonstrated the real possibility of utility of the stem cells in treatment of cleft lip and palate and encouraged us to move this research.
de Mendonça Costa et al5 in an experimental study demonstrated more mature bone formation using stem cells in rats cranial defects.
Bueno et al4 used orbicular oris muscle in rats and demonstrated reduction of granulation tissue and bone formation. Ramazanzadeh et al13 demonstrated, in an experimental study, the effectiveness of the stem cells in the orbicularis oral muscle function.
Brock et al12 did an experimental study using platelet-rich plasma and mesenchymal stem cell from adipose tissue and had more mature bone formation with the group that used bone fragments, platelet-rich plasma, and stem cels.
Spiekman et al14 reviewed the power of adipose-derived stromal cells in treatment of fibrotic scars trough effects in extracellular matrix remodeling, angiogenesis and modulation of inflammatory process. This study showed evidences of how the stem cells would benefit postoperative results.14
At the beginning of this study, it had a high cost to work with stem cells; however, after 10 years of the study it was noticed that the cost decreased 4-fold. Other negative aspect was the absence of other studies to be used as clinical guidelines to give us the assurance of no maleficence to the patients.
For these reasons it was opted for a limited number of patients in this study. The groups had only 9 patients each, a small sample number. This technique did not intend to cause any harm to the children and not interfere into the patient's craniofacial deformity treatment.
A long time of follow-up was defined to make sure that there will not be any patient maleficence and, second, to observe the clinical results in the soft and hard tissues operated.
At clinical researches the possibility of using stem cells in cleft lip and palate was cited initially by Hibi et al in alveolar cleft to restore bone. Other authors published good results with stem cells in the alveolar bone.7,15,16
Khojasteh et al16 in a literature review of the use of stem cell in alveolar cleft defects treatment concluded that there is no sufficient evidence of the treatment efficacy with tissue engineering, they talked about the necessity of well-designed controlled studies to be compared.
Ten years ago, when the present study started, the only evidence was that clinical trials with autologous stem cells did not cause damage to the patients and there was no rejection. However, it was not known if the outcomes could be advantageous to the patients.
The protocol used for collection, freezing, and thawing the stem cells followed the one from Brazil Cryogenic Center and this protocol was unable to supply our necessities because the protocols were made to a single use of the stem cells and in the cleft lip and palate treatment it is necessary to perform more than 1 surgical time. The protocol to stem cell storage to patients with cleft lip and palate was modified and the freezing was done into many storage bottles.
The plasticity of frozen stem cells is preserved (Matsuo et al), and this investigation suggested the possibility of using artificial bone grafting with the use of mesenchymal stem cells.17
The multiple storage in many bottles allowed us to use the stem cells in all surgical procedures and became pattern after the first case. Other problem was found with the stem cells injection.
The first use was done by direct infusion and it was observed that this option was good for the lip, but inappropriate for the palate, as the liquid with stem cells leaked through the palatal sutures.
The lip and palate require different techniques to apply stem cells: in the lip the direct infusion was appropriate. In the palate, it was first used hyaluronic acid as a matrix without success because the hyaluronic acid overflows from the surgical location. Second, the stem cell was mixed with washed powder collagen and it demonstrated to be a more efficient method to maintain the stem cells in the palate and to produce a hemostatic effect.
In these cases, with the use of stem cell into the posterior palate, there was no bone formation, but when the second surgery to close the hard palate was performed, it was observed by the surgeon that there was less amount of fibrotic tissue in all cases, particularly better in the last cases, which used a higher amount of stem cells and had a well-established protocol to use it in the palatal surgery.
The mean total nucleated hematopoietic cell number used was 19.92 × 106. It was used CD34 to express the stem cells. It is a glycosylated transmembrane protein, considered a marker for blood progenitor cell.
The mean number of stem cell used was 9.76 × 104. The ideal number of stem cells was not established yet and more researches will be necessary to do it. Actually, it is possible to amplify the number of the stem cells, technique that was impossible to be done in the present study but can be an option into future researches.
After the challenges of clinical injection of stem cells, it was needed to find clinical parameters to evaluate the results as described in the Method as it had not a histological parameter to demonstrate the tissue response to stem cell. The fibrous area between the soft and hard palate, which is usually resected in the anterior palatoplasty, could be useful to be sent for histological analyses and provide a more objective evidence of the inflammatory process in palatal tissue.
There were experimental studies with labeled stem cell with superparamagnetic iron oxide nanoparticles and visualization by magnetic resonance imaging (Jasmin et al).18 This technique could be able to demonstrate the stem cells into the surgical area, but was not possible to find commercial superparamagnetic iron oxide nanoparticles to be used in this study.
The result analysis was subjective with clinical examination and photos. Tomography was thought to be an appropriate examination to analyze the stem cell activity into bone cleft; however, it did not demonstrate any evidence of osseous development. For this reason this examination was performed only in 1 patient to avoid possible radiation consequences.
The subjective analysis demonstrated that the group of patients with stem cell injection had less inflammatory response at lip soft tissue, less scar hypertrophy, there was no palate fistula or dehiscence and less fibrosis between hard and soft palate at the second palatal surgery. However, there was no evidence of bone neoformation.
The statistical analysis demonstrated significant improvement of the inflammatory process of the lip in Stem Cell Group, different from the control group.
The width of the palate cleft was not different in the 2 groups. The use of palatal plates very early and a constant maintenance of these devices help an adequate palatal development and the use of stem cells could be a favorable factor to rush the process.
To develop bone tissue at the palate gap, it was noticed that only stem cells were not enough. It possibly may happen if the stem cells were associated with other growth factors or regenerative procedures. The use of Bone Morphogenetic Protein-2 and platelet-rich plasma could induce osseous neoformation and reduce the palate cleft.2,12 Another possibility of improving the results and performing bone formation could be the use of a higher number of stem cells. These possibilities open new pathways to future researches.
The inflammatory result, with less hypertrophy and dehiscence, was the most effective result comparing the 2 groups. The analysis demonstrated statistical difference with better tissue response into the group with stem cell.
This study was designed due to the desire of the authors to achieve better results in the cleft lip and palate treatment. The potential regenerative power of the stem cell stimulated and encouraged to find new methods to be added to the classical surgical techniques and make possible to reach better results with a small number and size of surgeries for cleft patient.
The development of protocols to collect, freeze, thaw, amplify, apply, and observe the outcomes will permit us to open a clinical research line with stem cells and cleft lip and palate surgery. It aims to improve results in soft and hard tissues, with less surgery in a near future, with other sources of stem cells that allow an increase of mesenchymal cells quantity.
At the beginning of the study, 10 years ago, it was not allowed to perform stem cells amplification by the Regulatory Health Institutions. Actually, it is possible to amplify the cells, which lead to a significant increasing number of the stem cells and consequently it should improve the clinical results of these cells use in cleft lip and palate patients.
Other experimental studies demonstrated the improvement of the stem cells in soft tissue including the muscle, this study over used it into subcutaneous and skin tissue achieving better muscular function, lip projection, and better scar appearance.
With these initial clinical observations and a defined clinical protocol, it will be able to proceed with researches looking for improvement at cleft lip and palate patient's results.
Our conclusion of this preliminary report is that stem cells in cleft lip and palate surgery decrease the inflammatory process and develop better scars than the regular series. The stem cells used in association with the regular surgical treatment do not cause any damage to the patient.
Besides the clinical conclusion, we developed a clinical protocol to safety use of stem cells, in a high amount of stem cells, with future possibilities to get better results in future clinical series.
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Keywords:© 2018 by Mutaz B. Habal, MD.
Cleft lip; cleft palate; reconstructive surgery; stem cell