|Year : 2018 | Volume
| Issue : 1 | Page : 13-18
Tenogenic differentiation of mesenchymal stem cells improves healing of linea alba incision
Dong Wang, Zhen-Ling Ji, Jing-Min Wang, Yu-Yan Tan
Department of General Surgery, School of Medicine, Zhongda Hospital, Southeast University, Nanjing 210009, Jiangsu, China
|Date of Submission||02-May-2018|
|Date of Acceptance||02-May-2018|
|Date of Web Publication||16-May-2018|
87 Ding Jia Qiao, Nanjing 210009, Jiangsu
Source of Support: None, Conflict of Interest: None
OBJECTIVE: The aim of this study is to investigate the curative effects of mesenchymal stem cells' (MSCs') tenogenic differentiation on Linea alba incision healing.
MATERIALS AND METHODS: Autologous MSCs were isolated from rat bone marrow and cultured and induced by 10 ng/mL of bone morphogenetic protein-12 (BMP-12) for 48 h. Expression of scleraxis (SCX), collagen I, and collagen III was examined at 48 h, 5 days, and 7 days to investigate the tenogenic differentiation. Fifty Sprague-Dawley rats were randomly divided into five groups: tenogenically differentiated (group E) or native mesenchymal stem cells (group D) seeded onto collagen sponge scaffolds or only sponge scaffolds (group C) were transplanted into the linea alba incision; rats that underwent operation without implantation of anything served as the sham group (group B), and rats that did not undergo operation were used as the control group (group A). Histological analysis was performed to explore the curative effects.
RESULTS: The expression of SCX increases continually even in the absence of BMP-12 for 5 days (P < 0.01). However, the expression of collagen I and III requires persistent inducing by BMP-12. Abundant numbers of cells are present in the midline incision compared to the native linea alba structure (Group A), and Group B has the most serious inflammation, with obvious inflammatory corpuscles. From the sections stained with Masson's trichrome, the tenogenic differentiation of MSCs treating the Linea alba incision demonstrates a relatively rich and well-aligned collagen fibrous matrix along the transverse (tensile) axis of the incision.
CONCLUSIONS: In animal experiments, MSCs' tenogenic differentiation induced by BMP-12 can dramatically enhance linea alba incision healing.
Keywords: Bone morphogenetic protein-12, linea alba incision, mesenchymal stem cells
|How to cite this article:|
Wang D, Ji ZL, Wang JM, Tan YY. Tenogenic differentiation of mesenchymal stem cells improves healing of linea alba incision. Int J Abdom Wall Hernia Surg 2018;1:13-8
| Introduction|| |
Incisional hernia is the most common complication following abdominal surgery. The reported incidence after midline laparotomy ranges from 3% to 26% and increases rapidly if the native operation is complicated by wound infection or other conditions., Despite extensive research on hernia repair, recurrent herniation continues to be constant. Compared to hernioplasty, enhancing the incision healing in the early stage plays a potentially more important role in preventing incisional hernia formation.
Recent tissue regeneration strategies aim to improve the outcome of incision healing. The linea alba consists of transverse and oblique collagen fibers , and cutting off these fibril bundles constitutes the anatomical basis of midline incisional hernia. Collagen regeneration and remolding, especially the mature form of type I collagen, is the most important event in the healing of the midline incision. Tendon fibroblasts build and maintain the extracellular matrix of tendons/ligaments and play an important role in this process. In addition to the local fibroblasts, mesenchymal stem cells (MSCs) also contribute to wound healing.
MSCs are nonhematopoietic progenitor cells that can differentiate into multiple cell lineages. Several studies have demonstrated that MSCs can differentiate into tenocyte-like fibroblasts in response to chemical factors, including bone morphogenetic proteins (BMPs)., The objective of this study is to investigate the curative effects of MSCs' tenogenic differentiation and transplantation on incision healing induced by BMP-12. To the best of our knowledge, this study is a novel exploration of MSCs' regenerative application in abdominal wall surgery.
| Materials and Methods|| |
Laboratory animals and groups
Fifty Sprague-Dawley rats (Silaike Inc., Shanghai, China), weighing between 150 g and 200 g, were randomly divided into five groups: Tenogenically differentiated MSCs (Group E) or native MSCs (Group D) seeded onto collagen sponge scaffolds (Yierkang Inc., Beijing, China) or only sponge scaffolds (Group C) were transplanted into the linea alba incision; rats that underwent operation without implantation of anything served as the sham group (Group B) and the rats that did not undergo any operation served as the control group (Group A).
Isolation and culture of mesenchymal stem cells
The rat bone marrow-derived MSCs were prepared as described., Briefly, bone marrow was collected by flushing the femur and tibia with medium and MSCs were isolated by gradient centrifugation for 30 min at room temperature, using Ficoll–Paque PLUS (1.077 g/mL, GE Healthcare Life Sciences). After centrifugation, cell pellets were resuspended in Dulbecco's Modified Eagle's Medium with low glucose (Invitrogen, USA), supplemented with 10% fetal bovine serum and incubated at 37°C in a humidified atmosphere containing 5% CO2 with regular replenishment of medium every 2–3 days.
Bone morphogenetic protein-12 inducing tenogenic differentiation of mesenchymal stem cells
MSCs at passage 3 were plated at a density of 5 × 102 cells/35 mm dish and treated with BMP-12 (Sigma-Aldrich, USA) of 10 ng/mL for 48 h. Then, BMP-12 was removed and replaced by normal medium without BMP-12 for another 5 days. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate the expression of scleraxis (SCX), collagen I, and collagen III at 48 h, 5 days, and 7 days. The gene-specific primers are presented in [Table 1]. The relative quantity of target gene expression was analyzed using the comparative CT (2−ΔΔCT) method. GAPDH was used as an endogenous reference gene.
|Table 1: Primers for reverse transcription polymerase chain reaction analysis|
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In vivo implantation
The MSCs–sponge complex was built as previously described. MSCs were suspended in growth medium at 1 × 106 cells/mL, and 0.5 mL was seeded onto sterilized 2 mm × 10 mm collagen sponge scaffolds. Cell-seeded scaffolds were placed in culture dishes and incubated for 2 h in a minimum volume of normal medium [Figure 1]a, after which more medium was applied to submerge the scaffolds for 24 h. Then, the MSCs–sponge complex was cultured for an additional 7 days using 10 ng/mL of BMP-12 or normal medium.
|Figure 1: In vivo implantation: (a) preparation of mesenchymal stem cells–sponge complex; (b) creation of linea alba incision; (c) closure of the incision with mesenchymal stem cells–sponge complex; (d) sample of healed linea alba incision|
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Animals were anesthetized with 0.3% pentobarbital sodium and the abdominal skin was sterilized with iodophor. A longitudinal midline skin incision was made to expose the linea alba and a 10-mm long defect was created along the linea alba [Figure 1]b, referring to a previous study. Then, the linea alba incision was closed using 5-0 prolene (Ethicon, USA) with or without the MSCs–sponge complex [Figure 1]c. The suture/incision ratio was 4:1.
After the operation, the animals were raised for another 4 weeks and then euthanized. The whole abdominal wall was obtained for further analysis [Figure 1]d. The sample including the linea alba scar was fixed in 10% neutral-buffered zinc–formalin for histological analysis.
The fixed tissues were cut transversely to present the healing of the linea alba scar and stained with hematoxylin and eosin and Masson's trichrome.
| Results|| |
Bone morphogenetic protein-12 induces mesenchymal stem cells tenocyte differentiation
SCX is predominantly expressed in tendons and is considered the most reliable phenotypic marker of the tenocytic lineage. The expression levels of SCX, collagen I, and collagen III at 48 h, 5 days, and 7 days are shown in [Figure 2]. Although BMP-12 was removed after 48 h, the expression of SCX continued to increase until 7 days (P < 0.01). Collagen I and collagen III show a reversal of the pattern, i.e., their expressions require persistent induction by BMP-12. The expression of collagen I is more sensitive than the expression of collagen III, with a rapid increase in collagen I at 48 h (P < 0.01). However, their expression is decreased when BMP-12 is removed from the medium.
|Figure 2: Bone morphogenetic protein-12 induces mesenchymal stem cells tenocyte differentiation: Increasing expression of scleraxis persists when bone morphogenetic protein-12 is removed. However, bone morphogenetic protein-12 has a decisive effect on collagen I and collagen III, i.e., their increased expression requires persistent induction by bone morphogenetic protein-12. *P < 0.05|
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Induced mesenchymal stem cells enhance collagen remodeling of linea alba incision
The tenogenic differentiation of MSCs can obviously enhance the healing of the linea alba incision, as shown in histological sections [Figure 3] and [Figure 4]. Of the four experimental groups [Figure 3]B1, [Figure 3]B2, [Figure 3]B3, [Figure 3]C1, [Figure 3]C2, [Figure 3]C3, [Figure 3]D1, [Figure 3]D2, [Figure 3]D3, [Figure 3]E1, [Figure 3]E2, [Figure 3]E3, abundant numbers of cells are present in the midline incision compared to the native linea alba structure [Figure 3]A1, [Figure 3]A2, [Figure 3]A3, and Group B has the most serious inflammation, with obvious inflammatory corpuscles [Figure 3]B1, [Figure 3]B2, [Figure 3]B3. From the sections of Masson's trichrome, the tenogenic differentiation of MSCs [Figure 4]E1, [Figure 4]E2, [Figure 4]E3 in treating the linea alba incision demonstrates the relatively rich and well-aligned collagen fibrous matrix along the transversal (tensile) axis of the incision. By contrast, the muscles and fibrous matrix are significantly disorderly organized in the tissues not treated by MSCs [Figure 4]B1, [Figure 4]B2, [Figure 4]B3, [Figure 4]C1, [Figure 4]C2, [Figure 4]C3.
|Figure 3: Hematoxylin and eosin staining: In the four experimental groups (B1-B3, C1-C3, D1-D3, E1-E3), abundant numbers of cells are present in the midline incision compared to the native linea alba structure (A1-A3). Group B shows the most serious inflammation. A1, B1, C1, D1, E1 ×40; A2, B2, C2, D2, E2 ×100; and A3, B3, C3, D3, E3 ×200; A4 shows the native linea alba structure|
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|Figure 4: Masson's trichrome staining: Group E demonstrates the relatively rich and well-aligned collagen fibrous matrix along the transversal (tensile) axis of the incision. By contrast, the muscles and fibrous matrix in Groups B and C are significantly disorderly in organization A1, B1, C1, D1, E1 ×40; A2, B2, C2, D2, E2 ×100; A3, B3, C3, D3, E3 ×200|
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| Discussion|| |
With the development of materials and techniques, substantial attention is currently being paid to hernioplasty. From the perspective of prevention, more work should be performed before hernia occurs. Some researchers have already performed some explorations in wound healing. The incision healing depends on the balance of collagen synthesis, remolding, and degradation. In some studies,,, doxycycline was administered to improve complicated wound healing and hernia repair strength with concomitant reduction of matrix metalloprotease levels, which degraded the extracellular matrix. For collagen synthesis, MSCs are widely used as seed cells in tissue regeneration medicine because of their easy gaining, quick proliferation, and multiple differentiating abilities. There have been few studies focusing on MSCs for incision healing. Stoff et al. injected MSCs into intracutaneous incisions, which significantly inhibited scar formation and increased the tensile strength of the skin wounds. Heffner et al. combined platelet-rich plasma, collagen, and MSCs to investigate the healing effect on midline laparotomy incisions. The results indicated a marked improvement in abdominal wall strength and histological evaluation confirmed increased vascularity and collagen abundance. That study was the only research involving MSCs and linea alba healing. In this study, we confirm that BMP-12 induction of tenogenic differentiation of MSCs enhances the healing of linea alba incisions, which is consistent with the previous results.
Regarding MSCs in herniorrhaphy, few studies have performed pilot explorations in this field. Gao et al. tried to analyze the coating ability of various meshes of MSCs, and four another articles investigated the curative effects of hernia repair with surgical mesh in combination with MSCs.,,, In short, the use of MSCs for regeneration is a novel field in abdominal wall surgery. As Tez and Kiliç indicated the stem cell origin theory of the inguinal hernia, many further works are needed on MSCs, fascia, prosthesis, heterogeneity, and herniorrhaphy.
Many studies have demonstrated that exogenous BMP-12 can induce MSCs to undergo tenogenic differentiation with increasing expression of special markers. However, this effect seems time dependent and thus is more important for downstream target genes. SCX is a tendon/ligament-specific transcription factor that is required for tenogenic differentiation.,, Our results reveal that BMP-12 dramatically increases SCX expression when present, which is consistent with other studies., However, the expression was previously reported to decrease when BMP-12 was absent. Conversely, expression continues to increase in our study, which may be attributable to the longer inducing time. The expression levels of collagen I and III begin decrease when MSCs are no longer stimulated by BMP-12. We also investigated the expression of BMP-12 and the results are in unbearable confusion. Thus, we cannot draw conclusions regarding the expression of endogenous BMP-12. In short, gene transfection of BMP-12 into MSCs seems to be a reliable method to continually induce the tenogenic differentiation of MSCs.
In this study, we use a collagen sponge as an implant scaffold. However, the collagen scaffold is an interference factor for exploring the curative effect of MSCs. Thus, Group C transplants with only the collagen sponge served as the sham group. There are two main reasons to involve the collagen sponge in this trial. First, MSCs require continuing induction by BMP-12 to undergo tenogenic differentiation, and collagen is a better carrier than buffer or hyaluronan paste. At 1, 7, and 14 days, approximately 80%, 45%, and 8%, respectively, of the initial dose of BMP-12 is retained at the implant sites. Second, a collagen sponge can provide a confined space scaffold for the MSCs. For the healing of midline laparotomy incisions, we only want to increase the production of collagen in the linea alba and a sponge scaffold provides solidity to prevent the diffusion of MSCs.
| Conclusions|| |
In animal experiments, MSCs' tenogenic differentiation induced by BMP-12 can dramatically enhance linea alba incision healing.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Sanders DL, Kingsnorth AN. The modern management of incisional hernias. BMJ 2012;344:e2843.
Henriksen NA, Sørensen LT, Jorgensen LN, Agren MS. Circulating levels of matrix metalloproteinases and tissue inhibitors of metalloproteinases in patients with incisional hernia. Wound Repair Regen 2013;21:661-6.
Axer H, Keyserlingk DG, Prescher A. Collagen fibers in linea alba and rectus sheaths. I. General scheme and morphological aspects. J Surg Res 2001;96:127-34.
Axer H, von Keyserlingk DG, Prescher A. Collagen fibers in linea alba and rectus sheaths. J Surg Res 2001;96:239-45.
Tharappel JC, Ramineni SK, Reynolds D, Puleo DA, Roth JS. Doxycycline impacts hernia repair outcomes. J Surg Res 2013;184:699-704.
Lee JY, Zhou Z, Taub PJ, Ramcharan M, Li Y, Akinbiyi T, et al.
BMP-12 treatment of adult mesenchymal stem cells in vitro
augments tendon-like tissue formation and defect repair in vivo
. PLoS One 2011;6:e17531.
Shen H, Gelberman RH, Silva MJ, Sakiyama-Elbert SE, Thomopoulos S. BMP12 induces tenogenic differentiation of adipose-derived stromal cells. PLoS One 2013;8:e77613.
Lennon DP, Caplan AI. Isolation of rat marrow-derived mesenchymal stem cells. Exp Hematol 2006;34:1606-7.
Cai F, Wu XT, Xie XH, Wang F, Hong X, Zhuang SY, et al.
Evaluation of intervertebral disc regeneration with implantation of bone marrow mesenchymal stem cells (BMSCs) using quantitative T2 mapping: A study in rabbits. Int Orthop 2015;39:149-59.
Harth KC, Broome AM, Jacobs MR, Blatnik JA, Zeinali F, Bajaksouzian S, et al.
Bacterial clearance of biologic grafts used in hernia repair: An experimental study. Surg Endosc 2011;25:2224-9.
Rice RD, Ayubi FS, Shaub ZJ, Parker DM, Armstrong PJ, Tsai JW, et al.
Comparison of Surgisis, AlloDerm, and Vicryl woven mesh grafts for abdominal wall defect repair in an animal model. Aesthetic Plast Surg 2010;34:290-6.
Franz MG. The biology of hernia formation. Surg Clin North Am 2008;88:1-15, vii.
Stechmiller J, Cowan L, Schultz G. The role of doxycycline as a matrix metalloproteinase inhibitor for the treatment of chronic wounds. Biol Res Nurs 2010;11:336-44.
Kessler MW, Barr J, Greenwald R, Lane LB, Dines JS, Dines DM, et al.
Enhancement of achilles tendon repair mediated by matrix metalloproteinase inhibition via systemic administration of doxycycline. J Orthop Res 2014;32:500-6.
Stoff A, Rivera AA, Sanjib Banerjee N, Moore ST, Michael Numnum T, Espinosa-de-Los-Monteros A, et al.
Promotion of incisional wound repair by human mesenchymal stem cell transplantation. Exp Dermatol 2009;18:362-9.
Heffner JJ, Holmes JW, Ferrari JP, Krontiris-Litowitz J, Marie H, Fagan DL, et al.
Bone marrow-derived mesenchymal stromal cells and platelet-rich plasma on a collagen matrix to improve fascial healing. Hernia 2012;16:677-87.
Gao Y, Liu LJ, Blatnik JA, Krpata DM, Anderson JM, Criss CN, et al.
Methodology of fibroblast and mesenchymal stem cell coating of surgical meshes: A pilot analysis. J Biomed Mater Res B Appl Biomater 2014;102:797-805.
Mestak O, Matouskova E, Spurkova Z, Benkova K, Vesely P, Mestak J, et al.
Mesenchymal stem cells seeded on cross-linked and noncross-linked acellular porcine dermal scaffolds for long-term full-thickness hernia repair in a small animal model. Artif Organs 2014;38:572-9.
Bogdan VG, Zafranskaya MM, Gain YM, Demidchik YE, Bagatka SS, Ivanchik GI, et al.
Modification of collagen formation by mesenchymal stem cells isolated from human adipose tissue in culture and after autotransplantation for abdominal hernia plasty. Bull Exp Biol Med 2013;156:152-5.
Dolce CJ, Stefanidis D, Keller JE, Walters KC, Newcomb WL, Heath JJ, et al.
Pushing the envelope in biomaterial research: Initial results of prosthetic coating with stem cells in a rat model. Surg Endosc 2010;24:2687-93.
Zhao Y, Zhang Z, Wang J, Yin P, Zhou J, Zhen M, et al.
Abdominal hernia repair with a decellularized dermal scaffold seeded with autologous bone marrow-derived mesenchymal stem cells. Artif Organs 2012;36:247-55.
Tez M, Kiliç YA. Stem cell origin theory of the inguinal hernia. Med Hypotheses 2006;66:1042-3.
Schweitzer R, Chyung JH, Murtaugh LC, Brent AE, Rosen V, Olson EN, et al.
Analysis of the tendon cell fate using scleraxis, a specific marker for tendons and ligaments. Development 2001;128:3855-66.
Shukunami C, Takimoto A, Oro M, Hiraki Y. Scleraxis positively regulates the expression of tenomodulin, a differentiation marker of tenocytes. Dev Biol 2006;298:234-47.
Docheva D, Hunziker EB, Fässler R, Brandau O. Tenomodulin is necessary for tenocyte proliferation and tendon maturation. Mol Cell Biol 2005;25:699-705.
Seeherman HJ, Archambault JM, Rodeo SA, Turner AS, Zekas L, D'Augusta D, et al.
RhBMP-12 accelerates healing of rotator cuff repairs in a sheep model. J Bone Joint Surg Am 2008;90:2206-19.
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