ORIGINAL ARTICLE
Consideration of Bone Regeneration Effect of Stem Cells: Comparison of Bone Regeneration Between Bone Marrow Stem Cells and Adipose-Derived Stem Cells Daniel Seungyoul Han, MD, PhD,* Hee Kyung Chang, MD, PhD,Þ Keun Ryoung Kim, DDS, MSD,þ and Sang Min Woo, MD§
Background: Much controversy exists as to how stem cells efficiently differentiate and regenerate. To research how stem cell origin affects optimal differentiation and regeneration, the authors collected stem cells from bone marrow and fat and compared amounts of bone regeneration from both groups of cells. Methods: This study used 16 New Zealand white rabbits raised in similar surroundings and conditions. After collecting stem cells from bone marrow and fat, osteoblast generation was induced. In each rabbit, 2 craniectomies (10 10 mm) were made into each rabbit’s calvarium, and 0.2 mL (1 106 cells/mL) of bone marrowYderived and adipose-derived stem cells were transplanted into each defect. After 3 and 5 weeks of transplantation, computed tomography was conducted. After 6 weeks, regenerated bone tissue was collected and measured for volume, and biopsy was performed. Results: Both bone marrowY and adipose-derived stem cells were effective in bone regeneration of the defect. Bone marrow stem cells demonstrated greater differentiation into osteoblasts, but there was no difference in the amount of measured regenerated bone volume after 6 weeks. Conclusions: Adipose-derived stem cells differentiate directly into osteoblasts less often than do bone marrowYderived stem cells. However, the total amount of regenerated bone is almost the same because of the effect of indirect bone regeneration. As adiposederived stem cells are easily accessible and have the potential to
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From the *Armed Forces Daegu Hospital, Daegu; †Department of Pathology, Kosin University Graduate School, Busan; ‡Department of Orthodontics, Prettymiso Dental Clinics; and §Department of General Surgery, Asan Medical Center, Seoul, South Korea. Received April 15, 2013. Accepted for publication August 26, 2013. Address correspondence and reprint requests to Dr. Daniel Seungyoul Han, Department of Plastic and Reconstructive Surgery, The Armed Forces Daegu Hospital, Eunho-ri Hayang-eup Gyeongsan-si Gyeongsangbuk-do, Republic of Korea; E-mail: [email protected] The authors report no conflicts of interest. Copyright * 2014 by Mutaz B. Habal, MD ISSN: 1049-2275 DOI: 10.1097/SCS.0000000000000378
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abundantly proliferate into mesenchymal cells, they could be an effective bone regeneration material. Key Words: Adipose-derived stem cell, bone marrow stem cell, bone regeneration (J Craniofac Surg 2014;25: 196Y201)
A
pproaches to treat extensive bone defects in tissue engineering began with bone substitute studies and are now currently focusing on the pluripotent stromal cellYbased studies. In particular, adult stem cells are being investigated most actively because their use avoids ethical dilemmas and because they can be used as a regeneration treatment for diverse tissues, including muscle, cartilage, bone, and fat. Initially, most adult stem cell studies focused on bone marrowYderived mesenchymal stem cells, because of the belief that adult stem cells have the greatest regeneration potential and are also the most plentiful in bone marrow. Thus, ways to bring the use of bone marrow stem cells into the clinical setting have been intensively studied. However, bone marrow stem cells can be obtained only through bone marrow biopsy. This procedure can produce severe pain, making it difficult to obtain the informed consent of patients. Furthermore, the number of stem cells produced from this procedure is limited, and several culture passages are needed to obtain a viable number of stem cells. As the cells’ characteristics change with increasing number of culture passages, unlimited proliferation is impossible.1 Accordingly, adipose-derived stem cells have been increasingly applied in the research and clinical setting, because fat tissue is more easily obtained than bone marrowY and adipose-derived stem cells and is much more abundant.2 Theoretically, every stem cell is pluripotent and able to differentiate into various tissues such as bone, cartilage, blood vessels, or nerve, depending on individual circumstances. Nevertheless, how the origin of bone marrow or fat tissue affects stem cells’ ability to differentiate and to continue to regenerate remains controversial. Accordingly, the author compared the bone regeneration of the bone marrow stem cells and of the adipose stem cells and thus aimed to research the difference in the regeneration capacity of bone according to the origin of the stem cells.
MATERIALS AND METHODS Raising of Experimental Animals All rabbits for this experiment were supplied from a single farm, raised by a single keeper. A total of 16 healthy New Zealand
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Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
The Journal of Craniofacial Surgery
& Volume 25, Number 1, January 2014
Bone Marrow & Adipose-Derived Stem Cells
When stem cells differentiate into osteoblasts, they produce abundant extracellular matrix, such as collagen, which mineralizes. By observing how much mineralization occurs, differentiation of stem cells from bone marrow and fat were compared indirectly (Fig. 1). Mineralization was observed by fluorescence microscopy by adding 10 mg/mL of calcein to the medium.
Experimental Procedure and Surgery Rabbits were placed into an experimental restraint and injected with an intramuscular anesthetic, a mixture of ketamine (35 mg/kg) and xylazine (0.2Y0.5 mg/kg). Additional intramuscular injections of ketamine (15 mg/kg) and xylazine (0.2 mg/kg) were given after 1 hour to continue anesthesia. Hair was removed from the rabbit’s head, and the area sterilized with povidone solution. A sterile drape was then placed around the surgical field. The procedure was performed using sterile technique. An incision was made in the midline between both ears and eyes, from anterior to posterior. Pericranium and scalp were dissected outward, exposing parietal and frontal bone. Using a vibration saw, craniectomies of 100 mm2 (10 10 mm) were made at 2 sites into both parietal and frontal bones. Each layer of craniectomized bone was discretely taken off with the bone cutter, with the weight, length, width, and thickness of the bone fragments measured to calculate its volume. The area of bone defect was fully cleansed with saline to prevent any bone regeneration effect from bone powder. In addition, a 0.5-mm-thick silastic sheet was applied above the exposed dura to prevent the influence of bone regeneration from the meninges.2 Bone marrowY and adipose-derived stem cells in the amount of 0.2 mL (1 106 cells/mL) were placed into the left and right defect site with fibrin glue, respectively (Fig. 2). Fibrin glue was added to act as stem cell scaffold.3 The reason we did not set the contrasted part separately in this experiment is because the results of previous researches already confirmed that adipose and bone marrow stem cells are effective in the regeneration of bone defects.4Y8 Depending on its position, the
FIGURE 1. Identification of mineralization by calcein staining. Bone marrow stem cells (A), adipose-derived stem cells (B), original magnification 1000.
white rabbits (3 kg, males) were each placed into an iron breeding cage (420 500 300 mm). Their environment was kept at a temperature of 22-C T 3-C, a relative humidity of 50% T 10%, and an illumination of 150 to 300 lux for 12 hours a day. The rabbits were allowed pellet feed and sterilized tap water (UV sterilizer, M600; Shanghai Dynamic Industry Co) ad libitum. The experiments were conducted after 2 weeks of acclimation to the changed surroundings to prevent stress that could have affected the experiments. A single researcher conducted every experiment in this study to reduce possible error.
Preparation of Mesenchymal Stem Cells Rabbit bone marrowY and adipose-derived stem cells were harvested from tibia and abdomen, respectively. Harvested cells were cultured in complete culture medium, and 1 106 cells/mL were collected through passaged culture of 6 to 7 times for bone marrow stem cells and 4 times for adipose-derived stem cells. Bone development and differentiation were induced in osteogenic medium, which included an >-minimal essential medium, 10% calf serum, 0.1 mM dexamethasone, 10 mM b-glycerol phosphate, and 50 mg/mL ascorbic acid, for 2 weeks.
FIGURE 2. Intraoperative photograph of rabbit calvaria with 2 craniectomies. In defect on the left, bone marrow stem cells with fibrin glue were applied. In defect on the right, adipose-derived stem cells with fibrin glue were applied.
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FIGURE 3. Three-dimensional cranial CT images of 2 calvarial defects. Preoperative state (A), after 3 weeks (B), and after 5 weeks (C)].
volume of the rabbit’s skull differs greatly, so comparing 3 groups was not adequate. To avoid bone regeneration by periosteum, the authors did not suture the periosteum closed and only sutured the scalp with 4-0 nylon.9 After surgery, treatments such as crystalloids or antibiotics were withheld, and the rabbits were raised in the same way before surgery.
Analysis of Bone Regeneration (1) Three-dimensional cranial computed tomography (CT) Using three-dimensional cranial CT, bone regeneration and changes were measured in the 16 rabbits at 3 and 5 weeks. (2) Gross observation and volume measurement After 6 weeks, left- and right-sided bone defects were again removed using a vibration saw and bone cutter. The weight, length, width, and thickness of regenerated bone were then measured. Based on the calculated volume of the bone fragments, precise regeneration amounts were compared. At this time, the area of the previously inserted 0.1 mm of silastic sheet was used as a gauge of the size of the initial bone defect. (3) Histological examination At 6 weeks, fragments were cut into a thickness of 5 Hm and stained with hematoxylin-eosin. Histological differences between specimens with bone marrowY and adipose-derived stem cells were observed under 40 and 200 magnification.
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FIGURE 4. Craniectomized bones almost completely regenerated by 6 weeks.
* 2014 Mutaz B. Habal, MD
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
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TABLE 1. Changes in Harvested Bone Volume After 6 Weeks
Pretreatment volume, mm2 Posttreatment volume, mm2 Regeneration ratio,† %
Bone Marrow Stem Cells
Adipose-Derived Stem Cells
P*
159.5 141.75 88.9
160.5 138.875 86.5
0.009
*Statistical significance among groups tested by Wilcoxon signed ranks test (PG 0.05). †Regeneration ratio calculated as a percentage of pretreatment and posttreatment volume.
Statistical Analysis SPSS version 17.0 (SPSS Inc, Chicago, IL) and the Wilcoxon signed ranks test were used for data analysis, with P G 0.05 considered significant.
RESULTS After adding calcein to the medium, the degree of differentiation from stem cells into osteoblasts was observed by fluorescence microscopy, revealing the reactions of stem cells harvested from both bone marrow and fat tissue. Thus, the authors were able to confirm that bone marrowY and adipose-derived stem cells had differentiated into osteoblasts in the osteogenic medium. In addition, bone marrow stem cells showed a greater amount of reaction than did adiposederived stem cells, leading the authors to conclude that bone marrow stem cells differentiated into osteoblasts at a greater rate than did the adipose-derived stem cells (Fig. 1). At 3 weeks, three-dimensional cranial CT revealed that the left-sided defect site had filled with regenerated bone, whereas rightsided defect site had not fully closed. Specifically, bone regeneration was more evident in the left-sided defect injected with bone marrow stem cells than in the right-sided defect injected with adipose-derived stem cells. However, on three-dimensional cranial CT at 5 weeks, bony closure was observed in both defect sites with an almost similar degree of bone regeneration. Thus, the authors were able to document a substantial decrease in the gap in bone regeneration between the 2 sites as compared with that of 3 weeks (Fig. 3). At 6 weeks, regenerated bone fragments from both sites were collected and analyzed. No differences were evident by gross observation. However, the volume of the left-sided defects that had been filled with bone marrow stem cells averaged 2.4% higher than the right-sided defects that had been filled with adipose-derived stem cells. Nevertheless, substantial bone regeneration was confirmed in both sides over the 6 weeks of the experiment (Fig. 4 and Table 1). In addition, osteoblasts were also found in both defects by histological analysis. This finding confirms active bone regeneration. Of note, some cells that differentiated into adipose tissue were observed in the defect injected with adipose-derived stem cells (Fig. 5 and 6).
Bone Marrow & Adipose-Derived Stem Cells
cells demonstrated a high rate of bone protein gene expression, producing osteocalcin and Runx2, during the induction period of osteoblast development. These findings are the same as the results of Hayashi et al,4 showing mesenchymal stem cells originating from bone marrow and periosteum had superior osteoblast development and differentiation ability compared with other stem cells. Zaminy et al11 also reported similar results when comparing bone marrow stem cells with adipose-derived stem cells using melatonin to stimulate osteoblast development and differentiation, with more apoptosis seen in adipose-derived stem cells. Im et al12 even questioned the potential of the adipose-derived stem cells to develop into osteoblasts and chondroblasts compared with the ability of bone marrow stem cells. Consequently, as in a report of Kim et al,5 one would expect bone marrow stem cell activity to be high in an environment of osteoblast development and adipose-derived stem cell activity to be higher in an environment of adipose cell development. The present study is significant because the comparison between the regeneration effect of bone marrow stem cells and adipose-derived stem cells was analyzed by quantifying the amount of differentiated and regenerated bone per unit volume, whereas previous reports only measured and compared the 2 cell lines’ degree of differentiation into mesenchymal cells. For the comparison made in the present study, stem cells from bone marrow and fat tissue were injected into bone defects of
DISCUSSION A number of reports comparing the osteoblast differentiation ability between bone marrow stem cells and adipose-derived stem cells have been reported. Rebelatto et al10 showed no difference in the differentiation ability of osteoblasts from bone marrowY or adipose-derived stem cells. Nonetheless, many other reports have demonstrated a higher differentiation ability of bone marrow stem cells. Shafiee et al1 insisted that bone marrow stem cells show a higher alkaline phosphatase activity and mineralization during their bone developmentYinducing period, compared with the lower activity of adipose-derived stem cells. In addition, bone marrow stem
FIGURE 5. Representative sections of hematoxylin-eosinYstained bone marrow stem cells from calvarial bone defects at 6 weeks (A, original magnification 40; B, original magnification 200).
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FIGURE 6. Representative sections of hematoxylin-eosinYstained adipose-derived stem cell from calvarial bone defects at 8 weeks (A, original magnification 40; B, original magnification 200).
rabbit calvaria. The authors then took three-dimensional cranial CT, histological, and volume measurements of the regenerated bone. In the analysis of bone regeneration, the left-sided defect injected with bone marrow stem cells showed 2.4% more regeneration when quantified by unit volume compared with the right-sided defect that was injected with adipose-derived stem cells. This result conflicts with previous reported studies that showed the superior ability of bone marrow stem cells to differentiate into osteoblasts or that there is no usefulness in using adipose-derived stem cells for bone regeneration substitutes because they do not readily differentiate into osteoblasts. In the present study, the authors were able to confirm the superior mineralization effect of bone marrow stem cells by calcein staining and to indirectly confirm their ability to differentiate into osteoblasts in osteogenic medium as in previous reports. However, no significant difference in bone regeneration was found between bone marrow stem cells and adipose-derived stem cells in healing rabbit calvarial defect areas in this study. Accordingly, the authors were able to confirm that the degree of stem cell differentiation and amount of bone regeneration are not directly proportional to each other through this experiment. Thus, one should consider the bone regeneration ability of adipose-derived stem cells as superior, when compared with their degree of differentiation, not necessarily that the bone regeneration ability of bone marrow stem cells was less than expected.
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Dudas et al6 had previously shown the bone regeneration effect by injecting adipose-derived stem cells into cranial defect areas of rabbits. Yoon et al7reported better bone regeneration than the control group using adipose-derived stem cells in murine cranial defects. Lendeckel et al8 reported effective regeneration results using only an adipose-derived stem cell injection in a 7-year-old girl having an extensive cranial defect caused by trauma. As these reports demonstrate, the bone regeneration ability of adipose-derived stem cells has been widely confirmed. This ability may be due to another external phenomenon that affects bone regeneration indirectly, other than a direct bone regeneration effect through stem cell differentiation into osteoblasts. According to recent studies, although alkaline phosphatase activity and mineralization of adipose-derived stem cells are lower than bone marrow stem cells, the expression of collagen type 1, osteocalcin, osteopontin, and BMP-2 (bone morphogenetic protein 2) is prominent in their undifferentiated state.1 In particular, Pekkarinen et al13 showed that BMP, one of the most robust bone regeneration factors, even on its own, can demonstrate a bone regeneration effect in extensive areas of bone defect. Furthermore, growth factors such as transforming growth factor and fibroblast growth factor also affect stem cell differentiation through various interactions with adipose-derived stem cells.14 In other words, the direct effects of adipose-derived stem cells caused by their differentiation into osteoblasts and the indirect effects of osteocalcin in the matrix of adipose-derived stem cells, bone development proteins such as osteopontin and BMP, and the various growth factors expressed during the stimulation of bone development are all involved in bone regeneration. Thus, although the direct bone regeneration effect of adipose-derived stem cells is relatively weak, the indirect bone regeneration effects of bone development protein and various growth factors can effectively compensate for that weakness (Fig. 7). This corresponds with authors’ other findings that showed superior bone regeneration when injecting adiposederived stem cells with demineralized bone matrix compared with injecting only adipose-derived stem cells or demineralized bone matrix. Furthermore, it has been suggested that there is a small gap in the time between when adipose-derived stem cells directly and indirectly foster bone regeneration in their environment. Evidence for this is the fact that the right-sided defect injected with adiposederived stem cells showed relatively week bone regeneration at 3 weeks, but a much smaller difference when compared with the leftsided defect at 5 weeks on three-dimensional CT. In other words, the authors were able to confirm that the initial effect of adiposederived stem cells differentiation into osteoblasts is less than that of bone marrow stem cells, confirming that there is a gap in the time of direct bone regeneration. However, final regeneration amounts
FIGURE 7. Bone regeneration by stem cells consists of both direct and indirect effects.
* 2014 Mutaz B. Habal, MD
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were almost the same because of additional indirect bone regeneration caused by the activation of bone generation proteins. Also, the fact that the regenerated bone fragment produced by injection of adipose-derived stem cells showed prominent matrix cells, such as adipose tissue, in histological examination can be seen as additional evidence for this idea. Stem cells from both bone marrow and fat tissue can be effective treatments for bone regeneration. In particular, if a large difference in bone regeneration is not critical for healing a bony defect, like the present study suggests, selecting adipose-derived stem cells for bone regeneration treatment is ideal. This is because adipose-derived stem cells have a greater ability to proliferate than do bone marrow stem cells, and they are easier to harvest and culture over long periods. Another advantage of adipose-derived stem cells includes their cost-effectiveness due to the short time required to amass enough cells for bone regeneration. Zhu et al15 reported adipose-derived stem cells sustain their pluripotent phenotype and continue to strongly proliferate despite 25 culture passages. Therefore, this ability would be helpful if the treatment of extensive bone defects required stem cells to be passed through several cultures.
CONCLUSIONS Significant bone regeneration was shown in both experimental conditions, injection of bone marrow stem cells and adiposederived stem cells into areas of bone defect in rabbits. Compared with areas injected with adipose-derived stem cells, areas injected with bone marrow stem cells demonstrated 2.4% higher bone regeneration volume. Thus, the experiment shows that adipose-derived stem cells have almost the same bone regeneration ability as do bone marrowYderived stem cells. This finding was quite different from the results of previous reports that insisted the bone cell development and differentiation ability of adipose-derived stem cells is relatively weak and that using adipose-derived stem cells is an ineffective way to regenerate bone. Therefore, adipose-derived stem cells should be considered as an effective bone regeneration material because of their low cost, easy accessibility, and continuous proliferation without degeneration despite many culture passages. This may not only be because of the direct bone regeneration effects of adipose-derived stem cells through their differentiation into osteoblasts, but also because of their indirect effect on bone regeneration. Further research is needed to clarify these indirect effects of adipose-derived stem cells on bone regeneration.
Bone Marrow & Adipose-Derived Stem Cells
REFERENCES 1. Shafiee A, Seyedjafari E, Soleimani M, et al. A comparison between osteogenic differentiation of human unrestricted somatic stem cells and mesenchymal stem cells from bone marrow and adipose tissue. Biotechnol Lett 2011;33:1257Y1264 2. Lee JH, Lee KH, Kim MH, et al. Possibility of undifferentiated human thigh adipose stem cells differentiating into functional hepatocytes. Arch Plast Surg 2012;39:593Y599 3. Singh K, Moyer H, Williams JK, et al. Fibrin glue: a scaffold for cellular-based therapy in a critical-sized defect. Ann Plast Surg 2011;66:301Y305 4. Hayashi O, Katsube Y, Hirose M, et al. Comparison of osteogenic ability of rat mesenchymal stem cells from bone marrow, periosteum, and adipose tissue. Calcif Tissue Int 2008;82:238Y247 5. Kim D, Monaco E, Maki A, et al. Morphologic and transcriptomic comparison of adipose- and bone-marrow-derived porcine stem cells cultured in alginate hydrogels. Cell Tissue Res 2010;341:359Y370 6. Dudas JR, Marra KG, Cooper GM, et al. The osteogenic potential of adipose-derived stem cells for the repair of rabbit calvarial defects. Ann Plast Surg 2006;56:543Y548 7. Yoon E, Dhar S, Chun DE, et al. In vivo osteogenic potential of human adipose-derived stem cells/poly lactide-co-glycolic acid constructs for bone regeneration in a rat critical-sized calvarial defect model. Tissue Eng 2007;13:619Y627 8. Lendeckel S, Jo¨dicke A, Christophis P, et al. Autologous stem cells (adipose) and fibrin glue used to treat widespread traumatic calvarial defects: case report. J Craniomaxillofac Surg 2004;32:370Y373 9. Gosain AK, Gosain SA, Sweeney WM, et al. Regulation of osteogenesis and survival within bone grafts to the calvaria: the effect of the dura versus the pericranium. Plast Reconstr Surg 2011;128:85Y94 10. Rebelatto CK, Aguiar AM, Moreta˜o MP, et al. A. Dissimilar differentiation of mesenchymal stem cells from bone marrow, umbilical cord blood, and adipose tissue. Exp Biol Med 2008;233:901Y913 11. Zaminy A, RagerdiKashani I, Barbarestani M, et al. Osteogenic differentiation of rat mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells: melatonin as a differentiation factor. Iran Biomed J 2008;12:133Y141 12. Im GI, Shin YW, Lee KB. Do adipose tissueYderived mesenchymal stem cells have the same osteogenic and chondrogenic potential as bone marrowYderived cells? Osteoarthritis Cartilage 2005;13:845Y853 13. Pekkarinen T, Ja¨msa¨ T, Ma¨a¨tta¨ M, et al. Reindeer BMP extract in the healing of critical-size bone defects in the radius of the rabbit. Acta Orthop 2006;77:952Y959 14. Kim U, Choi Y, Jung J. The effect of growth factors on osteogenic differentiation of adipose tissueYderived stromal cells. J Korean Oral Maxillofac Surg 2006;32:327Y333 15. Zhu Y, Liu T, Song K, et al. Adipose-derived stem cell: a better stem cell than BMSC. Cell Biochem Funct 2008;26:664Y675
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Consideration of Bone Regeneration Effect of Stem Cells: Comparison of Bone Regeneration Between Bone Marrow Stem Cells and Adipose-Derived Stem Cells Daniel Seungyoul Han, MD, PhD,* Hee Kyung Chang, MD, PhD,Þ Keun Ryoung Kim, DDS, MSD,þ and Sang Min Woo, MD§
Background: Much controversy exists as to how stem cells efficiently differentiate and regenerate. To research how stem cell origin affects optimal differentiation and regeneration, the authors collected stem cells from bone marrow and fat and compared amounts of bone regeneration from both groups of cells. Methods: This study used 16 New Zealand white rabbits raised in similar surroundings and conditions. After collecting stem cells from bone marrow and fat, osteoblast generation was induced. In each rabbit, 2 craniectomies (10 10 mm) were made into each rabbit’s calvarium, and 0.2 mL (1 106 cells/mL) of bone marrowYderived and adipose-derived stem cells were transplanted into each defect. After 3 and 5 weeks of transplantation, computed tomography was conducted. After 6 weeks, regenerated bone tissue was collected and measured for volume, and biopsy was performed. Results: Both bone marrowY and adipose-derived stem cells were effective in bone regeneration of the defect. Bone marrow stem cells demonstrated greater differentiation into osteoblasts, but there was no difference in the amount of measured regenerated bone volume after 6 weeks. Conclusions: Adipose-derived stem cells differentiate directly into osteoblasts less often than do bone marrowYderived stem cells. However, the total amount of regenerated bone is almost the same because of the effect of indirect bone regeneration. As adiposederived stem cells are easily accessible and have the potential to
What Is This Box? A QR Code is a matrix barcode readable by QR scanners, mobile phones with cameras, and smartphones. The QR Code links to the online version of the article.
From the *Armed Forces Daegu Hospital, Daegu; †Department of Pathology, Kosin University Graduate School, Busan; ‡Department of Orthodontics, Prettymiso Dental Clinics; and §Department of General Surgery, Asan Medical Center, Seoul, South Korea. Received April 15, 2013. Accepted for publication August 26, 2013. Address correspondence and reprint requests to Dr. Daniel Seungyoul Han, Department of Plastic and Reconstructive Surgery, The Armed Forces Daegu Hospital, Eunho-ri Hayang-eup Gyeongsan-si Gyeongsangbuk-do, Republic of Korea; E-mail: [email protected] The authors report no conflicts of interest. Copyright * 2014 by Mutaz B. Habal, MD ISSN: 1049-2275 DOI: 10.1097/SCS.0000000000000378
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abundantly proliferate into mesenchymal cells, they could be an effective bone regeneration material. Key Words: Adipose-derived stem cell, bone marrow stem cell, bone regeneration (J Craniofac Surg 2014;25: 196Y201)
A
pproaches to treat extensive bone defects in tissue engineering began with bone substitute studies and are now currently focusing on the pluripotent stromal cellYbased studies. In particular, adult stem cells are being investigated most actively because their use avoids ethical dilemmas and because they can be used as a regeneration treatment for diverse tissues, including muscle, cartilage, bone, and fat. Initially, most adult stem cell studies focused on bone marrowYderived mesenchymal stem cells, because of the belief that adult stem cells have the greatest regeneration potential and are also the most plentiful in bone marrow. Thus, ways to bring the use of bone marrow stem cells into the clinical setting have been intensively studied. However, bone marrow stem cells can be obtained only through bone marrow biopsy. This procedure can produce severe pain, making it difficult to obtain the informed consent of patients. Furthermore, the number of stem cells produced from this procedure is limited, and several culture passages are needed to obtain a viable number of stem cells. As the cells’ characteristics change with increasing number of culture passages, unlimited proliferation is impossible.1 Accordingly, adipose-derived stem cells have been increasingly applied in the research and clinical setting, because fat tissue is more easily obtained than bone marrowY and adipose-derived stem cells and is much more abundant.2 Theoretically, every stem cell is pluripotent and able to differentiate into various tissues such as bone, cartilage, blood vessels, or nerve, depending on individual circumstances. Nevertheless, how the origin of bone marrow or fat tissue affects stem cells’ ability to differentiate and to continue to regenerate remains controversial. Accordingly, the author compared the bone regeneration of the bone marrow stem cells and of the adipose stem cells and thus aimed to research the difference in the regeneration capacity of bone according to the origin of the stem cells.
MATERIALS AND METHODS Raising of Experimental Animals All rabbits for this experiment were supplied from a single farm, raised by a single keeper. A total of 16 healthy New Zealand
The Journal of Craniofacial Surgery
& Volume 25, Number 1, January 2014
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
The Journal of Craniofacial Surgery
& Volume 25, Number 1, January 2014
Bone Marrow & Adipose-Derived Stem Cells
When stem cells differentiate into osteoblasts, they produce abundant extracellular matrix, such as collagen, which mineralizes. By observing how much mineralization occurs, differentiation of stem cells from bone marrow and fat were compared indirectly (Fig. 1). Mineralization was observed by fluorescence microscopy by adding 10 mg/mL of calcein to the medium.
Experimental Procedure and Surgery Rabbits were placed into an experimental restraint and injected with an intramuscular anesthetic, a mixture of ketamine (35 mg/kg) and xylazine (0.2Y0.5 mg/kg). Additional intramuscular injections of ketamine (15 mg/kg) and xylazine (0.2 mg/kg) were given after 1 hour to continue anesthesia. Hair was removed from the rabbit’s head, and the area sterilized with povidone solution. A sterile drape was then placed around the surgical field. The procedure was performed using sterile technique. An incision was made in the midline between both ears and eyes, from anterior to posterior. Pericranium and scalp were dissected outward, exposing parietal and frontal bone. Using a vibration saw, craniectomies of 100 mm2 (10 10 mm) were made at 2 sites into both parietal and frontal bones. Each layer of craniectomized bone was discretely taken off with the bone cutter, with the weight, length, width, and thickness of the bone fragments measured to calculate its volume. The area of bone defect was fully cleansed with saline to prevent any bone regeneration effect from bone powder. In addition, a 0.5-mm-thick silastic sheet was applied above the exposed dura to prevent the influence of bone regeneration from the meninges.2 Bone marrowY and adipose-derived stem cells in the amount of 0.2 mL (1 106 cells/mL) were placed into the left and right defect site with fibrin glue, respectively (Fig. 2). Fibrin glue was added to act as stem cell scaffold.3 The reason we did not set the contrasted part separately in this experiment is because the results of previous researches already confirmed that adipose and bone marrow stem cells are effective in the regeneration of bone defects.4Y8 Depending on its position, the
FIGURE 1. Identification of mineralization by calcein staining. Bone marrow stem cells (A), adipose-derived stem cells (B), original magnification 1000.
white rabbits (3 kg, males) were each placed into an iron breeding cage (420 500 300 mm). Their environment was kept at a temperature of 22-C T 3-C, a relative humidity of 50% T 10%, and an illumination of 150 to 300 lux for 12 hours a day. The rabbits were allowed pellet feed and sterilized tap water (UV sterilizer, M600; Shanghai Dynamic Industry Co) ad libitum. The experiments were conducted after 2 weeks of acclimation to the changed surroundings to prevent stress that could have affected the experiments. A single researcher conducted every experiment in this study to reduce possible error.
Preparation of Mesenchymal Stem Cells Rabbit bone marrowY and adipose-derived stem cells were harvested from tibia and abdomen, respectively. Harvested cells were cultured in complete culture medium, and 1 106 cells/mL were collected through passaged culture of 6 to 7 times for bone marrow stem cells and 4 times for adipose-derived stem cells. Bone development and differentiation were induced in osteogenic medium, which included an >-minimal essential medium, 10% calf serum, 0.1 mM dexamethasone, 10 mM b-glycerol phosphate, and 50 mg/mL ascorbic acid, for 2 weeks.
FIGURE 2. Intraoperative photograph of rabbit calvaria with 2 craniectomies. In defect on the left, bone marrow stem cells with fibrin glue were applied. In defect on the right, adipose-derived stem cells with fibrin glue were applied.
* 2014 Mutaz B. Habal, MD
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FIGURE 3. Three-dimensional cranial CT images of 2 calvarial defects. Preoperative state (A), after 3 weeks (B), and after 5 weeks (C)].
volume of the rabbit’s skull differs greatly, so comparing 3 groups was not adequate. To avoid bone regeneration by periosteum, the authors did not suture the periosteum closed and only sutured the scalp with 4-0 nylon.9 After surgery, treatments such as crystalloids or antibiotics were withheld, and the rabbits were raised in the same way before surgery.
Analysis of Bone Regeneration (1) Three-dimensional cranial computed tomography (CT) Using three-dimensional cranial CT, bone regeneration and changes were measured in the 16 rabbits at 3 and 5 weeks. (2) Gross observation and volume measurement After 6 weeks, left- and right-sided bone defects were again removed using a vibration saw and bone cutter. The weight, length, width, and thickness of regenerated bone were then measured. Based on the calculated volume of the bone fragments, precise regeneration amounts were compared. At this time, the area of the previously inserted 0.1 mm of silastic sheet was used as a gauge of the size of the initial bone defect. (3) Histological examination At 6 weeks, fragments were cut into a thickness of 5 Hm and stained with hematoxylin-eosin. Histological differences between specimens with bone marrowY and adipose-derived stem cells were observed under 40 and 200 magnification.
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FIGURE 4. Craniectomized bones almost completely regenerated by 6 weeks.
* 2014 Mutaz B. Habal, MD
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
The Journal of Craniofacial Surgery
& Volume 25, Number 1, January 2014
TABLE 1. Changes in Harvested Bone Volume After 6 Weeks
Pretreatment volume, mm2 Posttreatment volume, mm2 Regeneration ratio,† %
Bone Marrow Stem Cells
Adipose-Derived Stem Cells
P*
159.5 141.75 88.9
160.5 138.875 86.5
0.009
*Statistical significance among groups tested by Wilcoxon signed ranks test (PG 0.05). †Regeneration ratio calculated as a percentage of pretreatment and posttreatment volume.
Statistical Analysis SPSS version 17.0 (SPSS Inc, Chicago, IL) and the Wilcoxon signed ranks test were used for data analysis, with P G 0.05 considered significant.
RESULTS After adding calcein to the medium, the degree of differentiation from stem cells into osteoblasts was observed by fluorescence microscopy, revealing the reactions of stem cells harvested from both bone marrow and fat tissue. Thus, the authors were able to confirm that bone marrowY and adipose-derived stem cells had differentiated into osteoblasts in the osteogenic medium. In addition, bone marrow stem cells showed a greater amount of reaction than did adiposederived stem cells, leading the authors to conclude that bone marrow stem cells differentiated into osteoblasts at a greater rate than did the adipose-derived stem cells (Fig. 1). At 3 weeks, three-dimensional cranial CT revealed that the left-sided defect site had filled with regenerated bone, whereas rightsided defect site had not fully closed. Specifically, bone regeneration was more evident in the left-sided defect injected with bone marrow stem cells than in the right-sided defect injected with adipose-derived stem cells. However, on three-dimensional cranial CT at 5 weeks, bony closure was observed in both defect sites with an almost similar degree of bone regeneration. Thus, the authors were able to document a substantial decrease in the gap in bone regeneration between the 2 sites as compared with that of 3 weeks (Fig. 3). At 6 weeks, regenerated bone fragments from both sites were collected and analyzed. No differences were evident by gross observation. However, the volume of the left-sided defects that had been filled with bone marrow stem cells averaged 2.4% higher than the right-sided defects that had been filled with adipose-derived stem cells. Nevertheless, substantial bone regeneration was confirmed in both sides over the 6 weeks of the experiment (Fig. 4 and Table 1). In addition, osteoblasts were also found in both defects by histological analysis. This finding confirms active bone regeneration. Of note, some cells that differentiated into adipose tissue were observed in the defect injected with adipose-derived stem cells (Fig. 5 and 6).
Bone Marrow & Adipose-Derived Stem Cells
cells demonstrated a high rate of bone protein gene expression, producing osteocalcin and Runx2, during the induction period of osteoblast development. These findings are the same as the results of Hayashi et al,4 showing mesenchymal stem cells originating from bone marrow and periosteum had superior osteoblast development and differentiation ability compared with other stem cells. Zaminy et al11 also reported similar results when comparing bone marrow stem cells with adipose-derived stem cells using melatonin to stimulate osteoblast development and differentiation, with more apoptosis seen in adipose-derived stem cells. Im et al12 even questioned the potential of the adipose-derived stem cells to develop into osteoblasts and chondroblasts compared with the ability of bone marrow stem cells. Consequently, as in a report of Kim et al,5 one would expect bone marrow stem cell activity to be high in an environment of osteoblast development and adipose-derived stem cell activity to be higher in an environment of adipose cell development. The present study is significant because the comparison between the regeneration effect of bone marrow stem cells and adipose-derived stem cells was analyzed by quantifying the amount of differentiated and regenerated bone per unit volume, whereas previous reports only measured and compared the 2 cell lines’ degree of differentiation into mesenchymal cells. For the comparison made in the present study, stem cells from bone marrow and fat tissue were injected into bone defects of
DISCUSSION A number of reports comparing the osteoblast differentiation ability between bone marrow stem cells and adipose-derived stem cells have been reported. Rebelatto et al10 showed no difference in the differentiation ability of osteoblasts from bone marrowY or adipose-derived stem cells. Nonetheless, many other reports have demonstrated a higher differentiation ability of bone marrow stem cells. Shafiee et al1 insisted that bone marrow stem cells show a higher alkaline phosphatase activity and mineralization during their bone developmentYinducing period, compared with the lower activity of adipose-derived stem cells. In addition, bone marrow stem
FIGURE 5. Representative sections of hematoxylin-eosinYstained bone marrow stem cells from calvarial bone defects at 6 weeks (A, original magnification 40; B, original magnification 200).
* 2014 Mutaz B. Habal, MD
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FIGURE 6. Representative sections of hematoxylin-eosinYstained adipose-derived stem cell from calvarial bone defects at 8 weeks (A, original magnification 40; B, original magnification 200).
rabbit calvaria. The authors then took three-dimensional cranial CT, histological, and volume measurements of the regenerated bone. In the analysis of bone regeneration, the left-sided defect injected with bone marrow stem cells showed 2.4% more regeneration when quantified by unit volume compared with the right-sided defect that was injected with adipose-derived stem cells. This result conflicts with previous reported studies that showed the superior ability of bone marrow stem cells to differentiate into osteoblasts or that there is no usefulness in using adipose-derived stem cells for bone regeneration substitutes because they do not readily differentiate into osteoblasts. In the present study, the authors were able to confirm the superior mineralization effect of bone marrow stem cells by calcein staining and to indirectly confirm their ability to differentiate into osteoblasts in osteogenic medium as in previous reports. However, no significant difference in bone regeneration was found between bone marrow stem cells and adipose-derived stem cells in healing rabbit calvarial defect areas in this study. Accordingly, the authors were able to confirm that the degree of stem cell differentiation and amount of bone regeneration are not directly proportional to each other through this experiment. Thus, one should consider the bone regeneration ability of adipose-derived stem cells as superior, when compared with their degree of differentiation, not necessarily that the bone regeneration ability of bone marrow stem cells was less than expected.
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Dudas et al6 had previously shown the bone regeneration effect by injecting adipose-derived stem cells into cranial defect areas of rabbits. Yoon et al7reported better bone regeneration than the control group using adipose-derived stem cells in murine cranial defects. Lendeckel et al8 reported effective regeneration results using only an adipose-derived stem cell injection in a 7-year-old girl having an extensive cranial defect caused by trauma. As these reports demonstrate, the bone regeneration ability of adipose-derived stem cells has been widely confirmed. This ability may be due to another external phenomenon that affects bone regeneration indirectly, other than a direct bone regeneration effect through stem cell differentiation into osteoblasts. According to recent studies, although alkaline phosphatase activity and mineralization of adipose-derived stem cells are lower than bone marrow stem cells, the expression of collagen type 1, osteocalcin, osteopontin, and BMP-2 (bone morphogenetic protein 2) is prominent in their undifferentiated state.1 In particular, Pekkarinen et al13 showed that BMP, one of the most robust bone regeneration factors, even on its own, can demonstrate a bone regeneration effect in extensive areas of bone defect. Furthermore, growth factors such as transforming growth factor and fibroblast growth factor also affect stem cell differentiation through various interactions with adipose-derived stem cells.14 In other words, the direct effects of adipose-derived stem cells caused by their differentiation into osteoblasts and the indirect effects of osteocalcin in the matrix of adipose-derived stem cells, bone development proteins such as osteopontin and BMP, and the various growth factors expressed during the stimulation of bone development are all involved in bone regeneration. Thus, although the direct bone regeneration effect of adipose-derived stem cells is relatively weak, the indirect bone regeneration effects of bone development protein and various growth factors can effectively compensate for that weakness (Fig. 7). This corresponds with authors’ other findings that showed superior bone regeneration when injecting adiposederived stem cells with demineralized bone matrix compared with injecting only adipose-derived stem cells or demineralized bone matrix. Furthermore, it has been suggested that there is a small gap in the time between when adipose-derived stem cells directly and indirectly foster bone regeneration in their environment. Evidence for this is the fact that the right-sided defect injected with adiposederived stem cells showed relatively week bone regeneration at 3 weeks, but a much smaller difference when compared with the leftsided defect at 5 weeks on three-dimensional CT. In other words, the authors were able to confirm that the initial effect of adiposederived stem cells differentiation into osteoblasts is less than that of bone marrow stem cells, confirming that there is a gap in the time of direct bone regeneration. However, final regeneration amounts
FIGURE 7. Bone regeneration by stem cells consists of both direct and indirect effects.
* 2014 Mutaz B. Habal, MD
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
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were almost the same because of additional indirect bone regeneration caused by the activation of bone generation proteins. Also, the fact that the regenerated bone fragment produced by injection of adipose-derived stem cells showed prominent matrix cells, such as adipose tissue, in histological examination can be seen as additional evidence for this idea. Stem cells from both bone marrow and fat tissue can be effective treatments for bone regeneration. In particular, if a large difference in bone regeneration is not critical for healing a bony defect, like the present study suggests, selecting adipose-derived stem cells for bone regeneration treatment is ideal. This is because adipose-derived stem cells have a greater ability to proliferate than do bone marrow stem cells, and they are easier to harvest and culture over long periods. Another advantage of adipose-derived stem cells includes their cost-effectiveness due to the short time required to amass enough cells for bone regeneration. Zhu et al15 reported adipose-derived stem cells sustain their pluripotent phenotype and continue to strongly proliferate despite 25 culture passages. Therefore, this ability would be helpful if the treatment of extensive bone defects required stem cells to be passed through several cultures.
CONCLUSIONS Significant bone regeneration was shown in both experimental conditions, injection of bone marrow stem cells and adiposederived stem cells into areas of bone defect in rabbits. Compared with areas injected with adipose-derived stem cells, areas injected with bone marrow stem cells demonstrated 2.4% higher bone regeneration volume. Thus, the experiment shows that adipose-derived stem cells have almost the same bone regeneration ability as do bone marrowYderived stem cells. This finding was quite different from the results of previous reports that insisted the bone cell development and differentiation ability of adipose-derived stem cells is relatively weak and that using adipose-derived stem cells is an ineffective way to regenerate bone. Therefore, adipose-derived stem cells should be considered as an effective bone regeneration material because of their low cost, easy accessibility, and continuous proliferation without degeneration despite many culture passages. This may not only be because of the direct bone regeneration effects of adipose-derived stem cells through their differentiation into osteoblasts, but also because of their indirect effect on bone regeneration. Further research is needed to clarify these indirect effects of adipose-derived stem cells on bone regeneration.
Bone Marrow & Adipose-Derived Stem Cells
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* 2014 Mutaz B. Habal, MD
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
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