- Email: [email protected]
The regeneration and augmentation of bone with injectable osteogenic cell sheet in a rat critical fracture healing model
Injury, Int. J. Care Injured 46 (2015) 1457–1464
Contents lists available at ScienceDirect
Injury journal homepage: www.elsevier.com/locate/injury
The regeneration and augmentation of bone with injectable osteogenic cell sheet in a rat critical fracture healing model Takamasa Shimizu a,*, Manabu Akahane b, Yusuke Morita c, Shohei Omokawa a, Kenichi Nakano a, Tsutomu Kira a, Tadanobu Onishi a, Yusuke Inagaki a, Akinori Okuda a, Kenji Kawate d, Yasuhito Tanaka a a
Department of Orthopedic Surgery, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan Department of Public Health, Health Management and Policy, Nara Medical University School of Medicine, Kashihara, Nara 634-8521, Japan c Department of Biomedical Engineering, Doshisha University, Kyotanabe, Kyoto 610-0394, Japan d Department of Artificial Joint and Regenerative Medicine, Nara Medical University, Kashihara, Nara 634-8522, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 February 2015 Received in revised form 18 April 2015 Accepted 26 April 2015
Limitations in the current treatment strategies make cases with compromised bone healing challenging clinical problems. Osteogenic cell sheets (OCSs), fabricated from rat bone marrow stromal cells (BMSCs), contain enriched osteoblasts and extracellular matrix. Here, we evaluated whether the minimally invasive percutaneous injection of OCSs without a scaffold could be used as a treatment to increase bone regeneration in a critical fracture healing model. Critical fracture healing model was created in the femora of 60 male Fischer 344 inbred rats using marrow ablation and periosteal removal. The rats were then randomly divided into two groups. Six hours after fracture, one group received an injection of OCSs (OCS group), while the second group was injected with phosphate-buffered saline (PBS) (control group). Fracture healing was evaluated using radiological, histological, micro-computed tomography (CT) and biomechanical analyses. The radiological and histological evaluations demonstrated enhanced bone regeneration in the OCS group compared with that in the control group. By 12 weeks, the hard callus had been remodelled via recorticalization in the OCS group. By contrast, no fracture union was found in the rats in the control group. Biomechanical testing revealed a significantly higher maximum bending load in the OCS group compared with that in the control group. The results of the present study demonstrate that the injection of entire OCSs can enhance bone regeneration and lead to bony union in a critical fracture healing model. Therefore, this procedure offers a minimally invasive technique to promote hard tissue reconstruction and, in particular, bone repair strategies for cases with compromised bone healing. ß 2015 Elsevier Ltd. All rights reserved.
Keywords: Fracture healing Injectable bone Osteogenic cell sheet Bone marrow stromal cell Extracellular matrix Bone tissue-engineering Nonunion Bone regeneration
Introduction Cases with compromised bone healing are a challenging clinical problem for orthopaedic surgeons. Although treatment options do exist for this challenging and recalcitrant clinical problem, they are complex and costly, and often multiple procedures are required for treatment. Fracture healing is normally a spontaneous sequence of events briefly summarised as the initial inflammation, then soft and hard callus formation, and finally bone remodelling [1], which involves the interactions between osteoprogenitor cells, growth
* Corresponding author. Tel.: +81 744 22 3051; fax: +81 744 25 6449. E-mail addresses: [email protected], [email protected] (T. Shimizu). http://dx.doi.org/10.1016/j.injury.2015.04.031 0020–1383/ß 2015 Elsevier Ltd. All rights reserved.
factors and the extracellular matrix (ECM) [2]. When this process does not occur, as in cases with compromised fracture healing or segmental bone defect, surgical intervention is required, and it is commonly combined with autologous grafting, which improves the local repair environment by providing osteoprogenitor cells, structural substrates and bone-inducing proteins [1]. Autologous bone grafting remains the gold standard for the treatment of cases with compromised bone healing [3]. However, it is an invasive, open surgical procedure that requires autologous bone to be harvested from an alternative site within the patient. Furthermore, the primary concern is that an open surgery performed in nonunion patients may further reduce the osteogenic potential of the progenitor cells at the fracture site. As such, minimally invasive surgical options are desirable to avoid damaging the local environment and inducing a local
1458
T. Shimizu et al. / Injury, Int. J. Care Injured 46 (2015) 1457–1464
inflammatory response in an already compromised bone healing site. The percutaneous injection of bone marrow has been suggested as one low-risk and inexpensive solution for the treatment of cases with compromised bone healing [4], and the success rate was reported to be relatively high in established nonunion cases [1]. However, the number and concentration of osteoprogenitor cells harvested using this method vary significantly between individuals [5,6], and fewer progenitor cells are present in marrow samples from older patients. An alternative strategy to promote bony reconstruction is the implantation of tissue-engineered bone (TEB) developed from bone marrow stromal cells (BMSCs). A recently described injectable, cell-based TEB provides a noninvasive solution in addition to the current treatment options [7–9] that have attracted considerable attention within the field of orthopaedics. However, the use of foreign biomaterial compounds often stimulates an immune response, leading to granulation tissue formation at injury sites and ultimately fibrous tissue in the place of bone [10]. Reducing the immune response generated by these implanted biomaterials is therefore of critical importance, and it indicates the need for materials that are intrinsically nonimmunogenic or those with properties that can be modified to prevent recognition by the immune system. A scaffold-free material made from cells fits these requirements. Interest in the concept of cell-sheet engineering for regenerative medicine applications has recently been increasing. This approach is gradually being established as a reliable alternative to traditional tissue engineering and regenerative medicine methods, namely biodegradable scaffolds used to create tissue substitutes and the injection of isolated cells [11]. When cultured cells are harvested as intact sheets including their deposited ECM, they can be easily attached to host tissues with minimal cell loss. This concept allows cells to be recovered within their own ECM as a sheet with cohesive cell–cell and cell–ECM interactions [12]. A custom-designed, temperature-responsive culture dish is required for successful harvest of the cell sheets. One advantage of using cultured cell sheets is that they eliminate the need for scaffolds, precluding the strong inflammatory responses that are typically induced when biodegradable scaffolds are broken down. Several studies have shown the potential of this technology for reconstitution of the cornea [13] and the myocardium [14], and in hepatocyte transplantation [15] and renal tube epithelial cell transfer [16]. Zhou et al. reported a method to harvest intact cell sheets from standard culture dishes using a cell scraper, which made the use of cell-sheet technology more convenient and widely applicable [17]. Our group demonstrated that osteogenic cell sheets (OCSs) fabricated from BMSCs with dexamethasone (Dex) and ascorbic acid phosphate (AscP) can form bone tissue without the need for a scaffold after transplantation [18]. OCSs derived from different cell sources have been used in the production of vascularised tissue-engineering constructs [9,19], for the regeneration of periodontitis defects [20] and to enhance tendon–bone healing [21]. In a recent study from our group, we demonstrated that an OCS can enhance bone formation in a rat nonunion model [22]. Further, the percutaneous injection of an entire OCS was previously reported by our group, and the OCS, injected into subcutaneous sites on the dorsal surface of rats, formed the bone in the absence of a scaffold [23]. However, whether this minimally invasive approach encourages hard tissue reconstruction at bony sites and induces fracture repair has not yet been tested. To this end, the aim of the present study was to determine whether the percutaneous injection of entire OCSs enhances bone regeneration in order to demonstrate a novel method for the minimally invasive treatment of cases with compromised bone healing.
Materials and methods Study design Sixty male Fischer 344 inbred rats were divided into two groups: OCS and control groups (n = 30 each). A femoral critical fracture healing model was created in each rat, and the rats were sutured. After 6 h, two sheets of OCS in 1-ml phosphate-buffered saline (PBS) (Gibco, Invitrogen, Carlsbad, CA, USA) were injected into the osteotomy site in rats in the OCS group. Rats in the control group received an injection of PBS only. Femora were harvested from each group at 4, 8 and 12 weeks after the injection. Fracture healing was evaluated with radiological and histological analyses at 4, 8 and 12 weeks (n = 4). Micro-computed tomography (CT) and biomechanical analyses were also performed at 4, 8 and 12 weeks (n = 6). Ten additional rats were used to prepare sufficient quantities for injection. This research protocol was approved by the Institutional Animal Care and Use Committee of Nara Medical University, following all appropriate guidelines. Critical fracture healing model of the femur We modified a rat femur critical fracture healing model described previously [22]. Briefly, 12-week-old male Fischer 344 inbred rats (approximately 280 g) were anaesthetised with 2% isoflurane. The operative site was shaved and prepared with ethanol. A 5-cm lateral incision was made on the hind limb parallel to the femoral shaft, extending from the femoral condyle to the proximal part of the femur. The midshaft of the femur was exposed by dividing the vastus lateralis and the biceps femoris muscles, and a fracture was created by transverse osteotomy with an oscillating mini saw. The femoral canal was reamed with an 18-G needle (Terumo, Tokyo, Japan), and it was irrigated with 20 ml of sterile saline so that the bone marrow was completely ablated. The periosteum was removed as much as possible from the proximal to the distal ends of the femur, and the muscle attached to the periosteum was also removed. A K-wire (0.8 mm in diameter) was then inserted into the medullary canal in a retrograde fashion with the use of a motor-driven drill. The K-wire was positioned within the proximal part of the femur, and the distal end was then cut close to the articular surface of the knee. The wound was then irrigated with 10 ml of sterile saline, and it was closed with a 4/0 nylon suture. Unprotected weight bearing was allowed immediately after the operation. Postoperative pain was managed by the subcutaneous administration of buprenorphine hydrochloride. Postoperative antibiotics were administrated by intramuscular injection of penicillin prophylactically. The rats were fed a standard maintenance diet, and they were provided water ad libitum. BMSCs preparation from bone marrow aspirates The method for BMSCs preparation has been reported previously [18,22,23]. Briefly, bone marrow was obtained from the femoral shafts of 7-week-old male Fischer 344 inbred rats. Both ends of the femur were cut at the epiphysis and the bone marrow was aspirated from the marrow cavity using 10 ml of standard culture medium expelled from a syringe using a 21-G needle. Standard culture medium consisted of minimal essential medium (MEM) (Nacalai Tesque Inc., Kyoto, Japan) containing 15% fetal bovine serum (FBS) (Gibco, Invitrogen, Carlsbad, CA, USA) and 1% antibiotics (10,000 U/ml penicillin and 10,000 mg/ml streptomycin, Nacalai Tesque Inc.). Harvested cells were then transferred into two T-75 flasks (Falcon, BD, NJ, USA) containing 15 ml of standard culture medium. Cell culture was maintained in 95% humidified atmosphere with 5% CO2 at 37 8C. After reaching confluence,
T. Shimizu et al. / Injury, Int. J. Care Injured 46 (2015) 1457–1464
cultured cells were released from the culture substratum using trypsin/ethylenediamine tetra-acetic acid (EDTA) (Gibco). Preparation for OCS The method of OCS preparation has been previously reported [18,22,23]. Briefly, BMSCs were released using trypsin/EDTA, seeded at a cell density of 1 104 cells/cm2 in a 10-cm dish (100 mm 20 mm; Falcon, BD Biosciences, Franklin Lakes, NJ, USA) and cultured in a standard medium with 10-nM Dex (Sigma– Aldrich, St. Louis, MO, USA) and 82-mg/ml AscP (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 14 days. The cells were rinsed twice with PBS, and they were lifted as an OCS using a cell scraper. Total number of cells in an OCS was calculated based on the amount of DNA, which was determined with a DNA Quantification Kit (Primary Cell, Hokkaido, Japan) according to the instructions provided by the manufacturer. DNA analysis was performed with Hoechst 33258 Fluorometry. Briefly, the lifted OCS was digested with 2 ml of buffer solution, and it was lysed by 15-min sonication in an ice bath (n = 6). The samples (50 ml) of digestion and standard were also mixed with 1 ml of buffer solution and added with 50-ml working solution for 10 min at room temperature. Fluorescence of the sample mixture was analysed by a spectrophotometer (458 nm). Based on the linear relationship between fluorescence value and standard sample, a conversion factor was gained to calculate the cell number in an OCS. Suspended post-differentiated BMSCs were also collected from an OCS using trypsin/EDTA, and they were used to generate a standard. Based on the standardization, a linear relationship was obtained between the optical density and the suspended cell number (correlation R2 = 0.9621). The calculated cell number contained in an OCS in a 10-cm dish was averaged as 3.1 0.7 106 cells. Samples for injection Approximately 6 h after the first surgery, the rats were again sedated. Samples, prepared mentioned above, in 1-ml PBS were injected percutaneously into the fracture site of rats in each groups under the X-ray fluoroscopy using a 1-ml syringe (JMS Co., Ltd., Tokyo, Japan) to which a 16-G needle (Terumo, Tokyo, Japan) was attached. The injection was carefully applied anteriorly and posteriorly to the osteotomy site. This procedure was sophisticated with a contrast medium under the X-ray fluoroscopy beforehand in the pilot study, and we confirmed that samples were absolutely injected at the osteotomy site. Quantitative reverse transcriptase-polymerase chain reaction analysis for OCS Gene expression levels of alkaline phosphatase (ALP), osteocalcin (OC), runt-related transcription factor 2 (Runx2), SP7 (osterix), bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor-A (VEGF-A) were measured to assess the in vitro osteogenic and angiogenic potential of the OCS. Briefly, cells from the OCS and BMSCs were cultured in six-well plates for 14 days in osteogenic and standard media, respectively. RNA was harvested using an RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s protocol, and it was converted to complementary DNA (cDNA), as previously described [22]. The messenger RNA (mRNA) expression levels were assessed using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) (ABI PRISM 7700 Sequence Detection System, Applied Biosystems, Foster City, CA, USA), using the respective primers and probe set purchased from Applied Biosystems: ALP:
1459
Rn 00564931 m1, OC: Rn01455285 g1, Runx2: Rn01512298 m1, SP7: Rn02769744 s1, BMP-2: Rn00567818 m1, VEGF-A: Rn01511602 m1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH): Rn99999916 s1. Thermal cycling conditions were 10 min at 95 8C for the activation of the universal mixture AmpliTaq gold polymerase, followed by 40 cycles of 15 s at 95 8C for denaturing and 1 min at 60 8C for annealing and extension. Target mRNA levels were compared after correcting to GAPDH mRNA levels, which was used to adjust for differences in the efficiency of reverse transcription between the samples. Macroscopic and histological examination of OCS To explore how the process of injection into the defect site would alter the integrity of the cell sheet, we performed an in vitro test where OCSs were harvested by cell scraping and then injected into 10-cm dishes using the same needle and syringe used for the in vivo injections. The OCSs before and after in vitro injection were fixed in 10% neutral-buffered formalin solution (Wako Pure Chemical Industries) for 2 days, embedded in paraffin, cut parallel across the middle of the sheet and stained with hematoxylin and eosin (H&E) for light microscopic observation. Radiological evaluation Radiographs were taken at 4, 8 and 12 weeks after surgery for rats in all three groups. Animals were placed in a prone position with both limbs fully abducted and externally rotated under anaesthesia. Fracture union was determined by the presence of bridging callus on two cortices. Micro-CT and biomechanical evaluation Both treated and contralateral femurs (intact femur) were harvested from six rats in each group at 4, 8 and 12 weeks, and they were processed for micro-CT and biomechanical evaluation. Micro-CT was performed using a SMX-160CTS micro-CT (Shimadzu, Kyoto, Japan). Intramedullary fixation pins were removed prior to micro-CT analysis. After scanning, the femurs were subjected to standardised three-point bending tests performed using a universal testing machine (EZ graph, Shimadzu). The cross-head speed was 10 mm/min, with the maximum bending load-to-rupture determined as the ultimate load. The relative ratio (percentage maximum bending load) of the treated femur to the contralateral femur was calculated in each group. Histological evaluation For histology at 4, 8 and 12 weeks, the femurs were harvested from two rats in each group, and they were fixed in 4% paraformaldehyde in 0.1-M phosphate buffer (Wako Pure Chemical Industries) for 48 h. Subsequently, each femur was decalcified with 10% EDTA (Wako Pure Chemical Industries) for approximately 3 weeks, and it was embedded in paraffin. Paraffin sections (3 mm thick) were cut and stained with H&E and toluidine blue for histological evaluation. Statistical analysis All values are expressed as the mean standard deviation (SD). Mann–Whitney U-test was performed for comparisons between two experimental groups. Data analysis was conducted using the Statistical Package for Social Science (SPSS) software package (version 17.0; SPSS Inc., Chicago, IL, USA). A probability value p < 0.05 was considered to denote statistical significance.
1460
T. Shimizu et al. / Injury, Int. J. Care Injured 46 (2015) 1457–1464
Results Macroscopic and histological examination of OCS The macroscopic appearance of OCS after injection was quite similar to before harvesting, as determined using the in vitro injection assay. After injection, we observed that the OCS returned to their original shape within a few minutes (Fig. 1A). A histological evaluation of the OCS showed that the sheets folded over themselves, resulting in thick layered, cell-based structures (Fig. 1B). qRT-PCR expression for markers of osteogenesis in OCS and BMSCs In the in vitro analysis, the relative mRNA expression of ALP, OC, Runx2, SP7 and BMP-2 was significantly greater in the OCS group compared with that in the BMSC group. However, the relative mRNA expression of VEGF-A was lower in the OCS group compared with that in the BMSC group (Fig. 2). Radiological evaluation The OCS group displayed abundant callus formation around the fracture site at 4 weeks after OCS injection. By 8 weeks, the callus bridged both ends of the fractured bone, and the cortical gap on both sides of the defect site disappeared. This was later followed by callus consolidation by 12 weeks. None of the surrounding soft tissues showed isolated ossification from the cortex for rats treated with OCS at 12 weeks. By comparison, the control group showed a lack of vigorous callus formation around the fracture sites at 4 weeks. Although a small amount of callus formation was observed along the periosteum away from the fracture site at 8 weeks, this never extended to bridge the fracture site. By 12 weeks, the ends of the fractured bone had become rounded, and they were resorbed, giving the appearance of typical established nonunion (Fig. 3).
decrease in the thickness of the woven bone. The hard callus within the woven bone had been remodelled into the configuration of the host cortical and/or the trabecular bone. At 12 weeks, fracture healing was observed in the OCS group, but not in the control groups (Fig. 4). Histological evaluation In the OCS group, the osteotomy site was covered with newly formed bone at 4 weeks, and bridging ossification was observed at 8 weeks. By contrast, the control group demonstrated a large gap at the fracture site at 4 weeks, and by 8 weeks, the control group showed a fibrous tissue interposition between the ends of the fractured bone, which had become rounded and showed evidence of bony resorption. At 12 weeks, hard callus formation was observed in the OCS group, characterised by the formation of mineralized bone matrix in the absence of a cartilaginous template and a repopulation of the bone marrow in the canal. However, the control group showed large numbers of chondrocytes and fibrous granulation tissue at the fracture site, which is consistent with the characteristics of atrophic nonunion and non-bridging endochondral ossification at that time point (Fig. 5). Biomechanical evaluation The maximum bending load in the experimental femurs was determined across the course of the study. In cases where the experimental femurs did not rupture in the three-point bending tests, we determined the provisional maximum bending load to be that required to rupture the intact contralateral femurs (deflection of 1.2 mm). The percentage maximum bending load in the OCS group was significantly higher than those in the control group after 8 weeks (OCS: 69.4 16.8%, control: 7.9 7.8%, respectively, at 12 weeks). The percentage maximum bending load in the control groups was uniformly low throughout the experimental period (Fig. 6).
Micro-CT evaluation
Discussion
At 4 weeks, the micro-CT analysis demonstrated substantial new bone formation around the fracture site in the OCS group as compared with the control groups. This new bone formation continued to be observed at 8 weeks. By 12 weeks, the newly formed bone had united the two ends of the fracture, and it had started to undergo remodelling, as indicated by the progressive
The results of the present study show that the OCSs maintained their original structure after they were scraped and were successfully transplanted via a needle without causing too much disruption to the integrity of the sheet. Furthermore, injecting the entire OCSs into the osteotomy site of a rat femoral critical fracture healing model as a minimally invasive technique for bone repair
[(Fig._1)TD$IG]
Fig. 1. Macroscopic appearance and histological evaluation of osteogenic cell sheets (OCS). OCS was harvested by scraping, and it was injected into 10-cm dishes using a 1-ml syringe and a 16-G needle and cultured for 24 h. (A) The OCS returned to their pre-injection size within a few minutes. (B) Histology revealed that the OCS retained their cellsheet structure after injection.
[(Fig._2)TD$IG]
T. Shimizu et al. / Injury, Int. J. Care Injured 46 (2015) 1457–1464
1461
Fig. 2. Quantitative real-time PCR expression for markers of osteogenesis in osteogenic cell sheet (OCS) and BMSCs. Comparison of the in vitro gene expression changes after 14 days in culture under osteogenic or standard culture conditions, respectively. Values represent the mean SD for n = 5; *p < 0.05 versus BMSCs. All data were normalised to GAPDH transcript levels. Abbreviations: Alkaline phosphatase (ALP), osteocalcin (OC), bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF)-A, runtrelated transcription factor-2 (Runx2) and osterix (SP7).
has the potential to augment bone regeneration. The histological findings demonstrated a statistically significant increase in bone formation in the OCS group at 4 weeks and the formation of a bridging callus at the osteotomy site within the first 8 weeks. By
contrast, chondrocytes were present in the control group as late as 12 weeks, and the presence of fibrous granulation tissue between the bone fragments was consistent with the characteristics of atrophic nonunion at the fracture site. These results indicated the
[(Fig._3)TD$IG]
Fig. 3. Representative radiographs of fractured sites at 2, 4, 6, 8 and 12 weeks in OCS and control groups. The OCS group displayed abundant callus around the fracture site at 4 weeks. Between 8 and 12 weeks, the callus bridged both fractured ends, with a reduced frequency of gaps between the cortical bones. At 4 weeks, an absence of vigorous callus formation was noted in the control group around the fracture sites. Although small callus formation was seen along the periosteum away from the fracture site at 8 weeks, this failed to bridge the fracture site by 12 weeks.
[(Fig._4)TD$IG]
1462
T. Shimizu et al. / Injury, Int. J. Care Injured 46 (2015) 1457–1464
Fig. 4. Three-dimensional micro-CT evaluation of the OCS and control groups. The OCS group displayed newly formed bone around the fracture site at 4 weeks. At 8 weeks, the newly formed bone united both ends of the fracture, and the woven bone was remodeled into cortical and/or trabecular bone. At 12 weeks, fracture healing was observed in the OCS group, but not in the control groups. Scale bar: 3 cm.
utility of the percutaneous injection of entire scaffold-free OCSs as a minimally invasive surgical procedure for the treatment of cases with compromised bone healing. Osteoprogenitor cells play an important role in fracture healing, and they are contained within the periosteum [24] and bone marrow [25]. Furthermore, the circulation and vasculature [26,27], as well as the surrounding local tissues [28], offer an additional source of osteoprogenitor cells that has been implicated in bone formation and healing. Because clinical patients often exhibit delayed healing with compromised host bone tissue at fracture sites with reduced numbers of osteoprogenitor cells in their bone marrow [29], we used a rat critical bone healing model involving bone marrow ablation and the removal of femora periosteum, as previously reported by Kaspers et al. [30]. Peters et al. stated that this model appeared suitable for analysing the regenerative potential of transplanted cells in an osteogenic progenitor cellfree environment [31]. Thus, the effect of OCSs in this model system was isolated to generating the callus, and it did not induce the recruitment of endogenous osteoprogenitor cells.
Recently, Ma et al. demonstrated that the local injection of the fragments of multicellular aggregates produced from an OCS enhanced bone healing compared with that obtained in a cell-only control group in a rabbit model of delayed healing [32]. However, in their method, the OCS was cut into small, approximately 1-mmdiameter fragments to pass through the needle. This process may have disrupted the ECM within the OCS, as well as resulting in a loss in cell number. Moreover, it is technically demanding to cut a cell sheet into small fragments of approximately 1 mm in diameter with a scalpel, and it is easy to clog an 18-G syringe needle with the small fragments during aspiration. The ECM is responsible for transmitting a wealth of chemical and mechanical signals that mediate key aspects of cellular physiology, including adhesion, migration, proliferation, differentiation and death, in addition to providing support for cellular tissues and physical sites for cellular attachment [33]. Cell sheet technology preserves tight junctions between adjacent cells, which may be essential for maintaining apical–basal cell polarity [11], and it is useful for the preservation of deposited ECM during in vitro culture for bone tissue
[(Fig._5)TD$IG]
T. Shimizu et al. / Injury, Int. J. Care Injured 46 (2015) 1457–1464
1463
Fig. 5. Histological sections stained with fast green/safranin O for the OCS and control groups. The union process in the OCS group was quite different from the control groups. In the OCS group, the osteotomy site was covered with newly formed bone and bridging ossification at 4 and 8 weeks. By contrast, fibrous tissue was interposed between the ends of the fractured bone in the control groups. At 12 weeks, hard callus formation without cartilaginous template was observed in the OCS group, but the presence of chondrocytes and fibrous granulation tissue was observed at the fracture site in the control groups. Black scale bar: 500 mm; blue scale bar: 100 mm.
engineering applications [17]. In fact, the storage and release of various growth factors by the ECM produced by osteoprogenitor cells have been previously demonstrated [34]. Compared with previous methods [32], the percutaneous injection of entire OCSs
[(Fig._6)TD$IG]
Fig. 6. Biomechanical evaluation. The percentage (%) maximum bending load was increased in the OCS group throughout the course of the study. After 8 weeks, the percentage maximum bending load in the OCS group was significantly higher than those in the control groups. Values represent the mean SD for n = 6; *p < 0.05 versus control.
appears to maintain cell–cell interactions, and thus it offers distinct advantages for bone formation and regeneration. Therefore, the method described here improves and simplifies the previously reported methods, and it is an attractive, minimally invasive option that can be applied for the treatment of cases with compromised bone healing while maintaining the complex interplay of cells, growth factors and ECM. There are several limitations to this study. First, we used rat BMSCs. To translate this method into the clinic, we will need to assess the osteogenic potential of human OCSs for the treatment of cases with compromised bone healing. Second, we injected OCSs into the fracture site of the femur on the same day as when the osteotomy was created. However, in the clinical setting, the surgeon cannot use OCSs immediately during the initial operation because the OCS preparation requires several weeks of the BMSC expansion in culture. In practice, a surgeon would harvest the patient’s BMSCs during the initial operation for fracture treatment to create OCSs for use in later clinical cases. Future studies could elucidate the effects of the injection in a delayed union or in an established nonunion model using a variety of injection protocols, such as single versus multiple
1464
T. Shimizu et al. / Injury, Int. J. Care Injured 46 (2015) 1457–1464
injections. Third, other transplantation methods, besides the use of a 16-G needle as reported here, should be investigated. For example, Maeda et al. recently developed a device that allows the minimally invasive endoscopic transplantation of cell sheets fabricated in thermoresponsive dishes for the treatment of lung injury [35]. OCSs may also be applied for endoscopy-assisted bone reconstruction via small incisions. Conclusion Our results clearly demonstrate that the percutaneous injection of intact OCSs provides a minimally invasive technique to augment osteogenesis in cases with compromised bone healing. This minimally invasive technique therefore offers a promising approach for hard tissue reconstruction in the compromised bone healing situation. Not only for preventing nonunion or delayed union on high-risk patients, but our method could also be applied for several clinical applications, including augmentation of bone regeneration in segmental bone defects, osteonecrosis and distraction osteogenesis. Further experiments are necessary to validate the utility of this technique. Conflicts of interest statement There are no competing financial interests. Acknowledgements We thank Y. Kayukawa and N. Nitta (Doshisha University) for performing the micro-CT and the biomechanical evaluation, and F. Kunda, M. Yoshimura and M. Matsumura (Nara Medical University School of Medicine) for technical assistance. We also thank Y. Dohi, T. Ueha and A. Nakamura (Nara Medical University) for experimental advice. This work was supported by[1_TD$IF] [2_TD$IF]JSPS [3_TD$IF]KAKENHI Grant [4_TD$IF]Number 23592198. Grant support was also received from Japan Orthopaedics and Traumatology[5_TD$IF] Foundation, Inc. References [1] Pountos I, Georgouli T, George K, Giannoudis PV. Efficacy of minimally invasive techniques for enhancement of fracture healing: evidence today. Int Orthop 2010;34(1):3–12. [2] Schindeler A, McDonald MM, Bokko P, Little DG. Bone remodeling during fracture repair: the cellular picture. Semin Cell Dev Biol 2008;19(5):459–66. [3] Myeroff C, Archdeacon M. Autologous bone graft: donor sites and techniques. J Bone Joint Surg Am 2011;93(23):2227–36. [4] Hernigou P, Poignard A, Beaujean F, Rouard H. Percutaneous autologous bonemarrow grafting for nonunions. Influence of the number and concentration of progenitor cells. J Bone Joint Surg Am 2005;87(7):1430–7. [5] Muschler GF, Boehm C, Easley K. Aspiration to obtain osteoblast progenitor cells from human bone marrow: the influence of aspiration volume. J Bone Joint Surg Am 1997;79(11):1699–709. [6] Muschler GF, Nitto H, Boehm CA, Easley KA. Age- and gender-related changes in the cellularity of human bone marrow and the prevalence of osteoblastic progenitors. J Orthop Res 2001;19(1):117–25. [7] Xia Y, Mei F, Duan Y, Gao Y, Xiong Z, Zhang T, et al. Bone tissue engineering using bone marrow stromal cells and an injectable sodium alginate/gelatin scaffold. J Biomed Mater Res A 2012;100(4):1044–50. [8] Yamada Y, Nakamura S, Ito K, Kohgo T, Hibi H, Nagasaka T, et al. Injectable tissue-engineered bone using autogenous bone marrow-derived stromal cells for maxillary sinus augmentation: clinical application report from a 2–6-year follow-up. Tissue Eng Part A 2008;14(10):1699–707. [9] Zhao L, Tang M, Weir MD, Detamore MS, Xu HH. Osteogenic media and rhBMP2-induced differentiation of umbilical cord mesenchymal stem cells encapsulated in alginate microbeads and integrated in an injectable calcium phosphate–chitosan fibrous scaffold. Tissue Eng Part A 2011;17(7–8):969–79. [10] Anderson JM, Rodriguez A, Chang DT. Foreign body reaction to biomaterials. Semin Immunol 2008;20(2):86–100.
[11] Yang J, Yamato M, Kohno C, Nishimoto A, Sekine H, Fukai F, et al. Cell sheet engineering: recreating tissues without biodegradable scaffolds. Biomaterials 2005;26(33):6415–22. [12] Okano T, Yamada N, Sakai H, Sakurai Y. A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly (N-isopropylacrylamide). J Biomed Mater Res 1993;27(10):1243–51. [13] Nishida K, Yamato M, Hayashida Y, Watanabe K, Maeda N, Watanabe H, et al. Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface. Transplantation 2004;77(3):379–85. [14] Shimizu T, Yamato M, Isoi Y, Akutsu T, Setomaru T, Abe K, et al. Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces. Circ Res 2002;90(3):e40. [15] Kano K, Yamato M, Okano T. Ectopic transplantation of hepatocyte sheets fabricated with temperature-responsive culture dishes. Hepatol Res 2008;38(11):1140–7. [16] Kushida A, Yamato M, Isoi Y, Kikuchi A, Okano T. A noninvasive transfer system for polarized renal tubule epithelial cell sheets using temperature-responsive culture dishes. Eur Cell Mater 2005;10:23–30. [17] Zhou Y, Chen F, Ho ST, Woodruff MA, Lim TM, Hutmacher DW. Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts. Biomaterials 2007;28(5):814–24. [18] Akahane M, Nakamura A, Ohgushi H, Shigematsu H, Dohi Y, Takakura Y. Osteogenic matrix sheet-cell transplantation using osteoblastic cell sheet resulted in bone formation without scaffold at an ectopic site. J Tissue Eng Regen Med 2008;2(4):196–201. [19] Mendes LF, Pirraco RP, Szymczyk W, Frias AM, Santos TC, Reis RL, et al. Perivascular-like cells contribute to the stability of the vascular network of osteogenic tissue formed from cell sheet-based constructs. PLoS One 2012;7(7):e41051. [20] Uematsu K, Kawase T, Nagata M, Suzuki K, Okuda K, Yoshie H, et al. Tissue culture of human alveolar periosteal sheets using a stem-cell culture medium (MesenPRO-RSTM): in vitro expansion of CD146-positive cells and concomitant upregulation of osteogenic potential in vivo. Stem Cell Res 2013;10(1):1–19. [21] Chang CH, Chen CH, Liu HW, Whu SW, Chen SH, Tsai CL, et al. Bioengineered periosteal progenitor cell sheets to enhance tendon-bone healing in a bone tunnel. Biomed J 2012;35(6):473–80. [22] Nakamura A, Akahane M, Shigematsu H, Tadokoro M, Morita Y, Ohgushi H, et al. Cell sheet transplantation of cultured mesenchymal stem cells enhances bone formation in a rat nonunion model. Bone 2010;46(2):418–24. [23] Akahane M, Shigematsu H, Tadokoro M, Ueha T, Matsumoto T, Tohma Y, et al. Scaffold-free cell sheet injection results in bone formation. J Tissue Eng Regen Med 2010;4(5):404–11. [24] Hutmacher DW, Sittinger M. Periosteal cells in bone tissue engineering. Tissue Eng 2003;9(Suppl 1):S45–64. [25] Colnot C, Huang S, Helms J. Analyzing the cellular contribution of bone marrow to fracture healing using bone marrow transplantation in mice. Biochem Biophys Res Commun 2006;350(3):557–61. [26] Collett GD, Canfield AE. Angiogenesis and pericytes in the initiation of ectopic calcification. Circ Res 2005;96(9):930–8. [27] Eghbali-Fatourechi GZ, Lamsam J, Fraser D, Nagel D, Riggs BL, Khosla S. Circulating osteoblast-lineage cells in humans. N Engl J Med 2005;352(19): 1959–66. [28] Rumi MN, Deol GS, Singapuri KP, Pellegrini Jr VD. The origin of osteoprogenitor cells responsible for heterotopic ossification following hip surgery: an animal model in the rabbit. J Orthop Res 2005;23(1):34–40. [29] Sethe S, Scutt A, Stolzing A. Aging of mesenchymal stem cells. Ageing Res Rev 2008;5(1):91–116. [30] Kaspar K, Matziolis G, Strube P, Senturk U, Dormann S, Bail HJ, et al. A new animal model for boneatrophic nonunion: fixation by external fixator. J Orthop Res 2008;26(12):1649–55. [31] Peters A, Toben D, Lienau J, Schell H, Bail HJ, Matziolis G, et al. Locally applied osteogenic predifferentiated progenitor cells are more effective than undifferentiated mesenchymal stem cells in the treatment of delayed bone healing. Tissue Eng Part A 2009;15(10):2947–54. [32] Ma D, Zhong C, Yao H, Liu Y, Chen F, Li J, et al. Engineering injectable bone using bone marrow stromal cell aggregates. Stem Cells Dev 2011;20(6): 989–99. [33] Nelson CM, Bissell MJ. Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. Annu Rev Cell Dev Biol 2006;22:287–309. [34] Dallas SL, Rosser JL, Mundy GR, Bonewald LF. Proteolysis of latent transforming growth factor-beta (TGF-beta)-binding protein-1 by osteoclasts. A cellular mechanism for release of TGF-beta from bone matrix. J Biol Chem 2002;277(24): 21352–60. [35] Maeda M, Yamato M, Kanzaki M, Iseki H, Okano T. Thoracoscopic cell sheet transplantation with a novel device. J Tissue Eng Regen Med 2009;3(4):255–9.
Contents lists available at ScienceDirect
Injury journal homepage: www.elsevier.com/locate/injury
The regeneration and augmentation of bone with injectable osteogenic cell sheet in a rat critical fracture healing model Takamasa Shimizu a,*, Manabu Akahane b, Yusuke Morita c, Shohei Omokawa a, Kenichi Nakano a, Tsutomu Kira a, Tadanobu Onishi a, Yusuke Inagaki a, Akinori Okuda a, Kenji Kawate d, Yasuhito Tanaka a a
Department of Orthopedic Surgery, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan Department of Public Health, Health Management and Policy, Nara Medical University School of Medicine, Kashihara, Nara 634-8521, Japan c Department of Biomedical Engineering, Doshisha University, Kyotanabe, Kyoto 610-0394, Japan d Department of Artificial Joint and Regenerative Medicine, Nara Medical University, Kashihara, Nara 634-8522, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 February 2015 Received in revised form 18 April 2015 Accepted 26 April 2015
Limitations in the current treatment strategies make cases with compromised bone healing challenging clinical problems. Osteogenic cell sheets (OCSs), fabricated from rat bone marrow stromal cells (BMSCs), contain enriched osteoblasts and extracellular matrix. Here, we evaluated whether the minimally invasive percutaneous injection of OCSs without a scaffold could be used as a treatment to increase bone regeneration in a critical fracture healing model. Critical fracture healing model was created in the femora of 60 male Fischer 344 inbred rats using marrow ablation and periosteal removal. The rats were then randomly divided into two groups. Six hours after fracture, one group received an injection of OCSs (OCS group), while the second group was injected with phosphate-buffered saline (PBS) (control group). Fracture healing was evaluated using radiological, histological, micro-computed tomography (CT) and biomechanical analyses. The radiological and histological evaluations demonstrated enhanced bone regeneration in the OCS group compared with that in the control group. By 12 weeks, the hard callus had been remodelled via recorticalization in the OCS group. By contrast, no fracture union was found in the rats in the control group. Biomechanical testing revealed a significantly higher maximum bending load in the OCS group compared with that in the control group. The results of the present study demonstrate that the injection of entire OCSs can enhance bone regeneration and lead to bony union in a critical fracture healing model. Therefore, this procedure offers a minimally invasive technique to promote hard tissue reconstruction and, in particular, bone repair strategies for cases with compromised bone healing. ß 2015 Elsevier Ltd. All rights reserved.
Keywords: Fracture healing Injectable bone Osteogenic cell sheet Bone marrow stromal cell Extracellular matrix Bone tissue-engineering Nonunion Bone regeneration
Introduction Cases with compromised bone healing are a challenging clinical problem for orthopaedic surgeons. Although treatment options do exist for this challenging and recalcitrant clinical problem, they are complex and costly, and often multiple procedures are required for treatment. Fracture healing is normally a spontaneous sequence of events briefly summarised as the initial inflammation, then soft and hard callus formation, and finally bone remodelling [1], which involves the interactions between osteoprogenitor cells, growth
* Corresponding author. Tel.: +81 744 22 3051; fax: +81 744 25 6449. E-mail addresses: [email protected], [email protected] (T. Shimizu). http://dx.doi.org/10.1016/j.injury.2015.04.031 0020–1383/ß 2015 Elsevier Ltd. All rights reserved.
factors and the extracellular matrix (ECM) [2]. When this process does not occur, as in cases with compromised fracture healing or segmental bone defect, surgical intervention is required, and it is commonly combined with autologous grafting, which improves the local repair environment by providing osteoprogenitor cells, structural substrates and bone-inducing proteins [1]. Autologous bone grafting remains the gold standard for the treatment of cases with compromised bone healing [3]. However, it is an invasive, open surgical procedure that requires autologous bone to be harvested from an alternative site within the patient. Furthermore, the primary concern is that an open surgery performed in nonunion patients may further reduce the osteogenic potential of the progenitor cells at the fracture site. As such, minimally invasive surgical options are desirable to avoid damaging the local environment and inducing a local
1458
T. Shimizu et al. / Injury, Int. J. Care Injured 46 (2015) 1457–1464
inflammatory response in an already compromised bone healing site. The percutaneous injection of bone marrow has been suggested as one low-risk and inexpensive solution for the treatment of cases with compromised bone healing [4], and the success rate was reported to be relatively high in established nonunion cases [1]. However, the number and concentration of osteoprogenitor cells harvested using this method vary significantly between individuals [5,6], and fewer progenitor cells are present in marrow samples from older patients. An alternative strategy to promote bony reconstruction is the implantation of tissue-engineered bone (TEB) developed from bone marrow stromal cells (BMSCs). A recently described injectable, cell-based TEB provides a noninvasive solution in addition to the current treatment options [7–9] that have attracted considerable attention within the field of orthopaedics. However, the use of foreign biomaterial compounds often stimulates an immune response, leading to granulation tissue formation at injury sites and ultimately fibrous tissue in the place of bone [10]. Reducing the immune response generated by these implanted biomaterials is therefore of critical importance, and it indicates the need for materials that are intrinsically nonimmunogenic or those with properties that can be modified to prevent recognition by the immune system. A scaffold-free material made from cells fits these requirements. Interest in the concept of cell-sheet engineering for regenerative medicine applications has recently been increasing. This approach is gradually being established as a reliable alternative to traditional tissue engineering and regenerative medicine methods, namely biodegradable scaffolds used to create tissue substitutes and the injection of isolated cells [11]. When cultured cells are harvested as intact sheets including their deposited ECM, they can be easily attached to host tissues with minimal cell loss. This concept allows cells to be recovered within their own ECM as a sheet with cohesive cell–cell and cell–ECM interactions [12]. A custom-designed, temperature-responsive culture dish is required for successful harvest of the cell sheets. One advantage of using cultured cell sheets is that they eliminate the need for scaffolds, precluding the strong inflammatory responses that are typically induced when biodegradable scaffolds are broken down. Several studies have shown the potential of this technology for reconstitution of the cornea [13] and the myocardium [14], and in hepatocyte transplantation [15] and renal tube epithelial cell transfer [16]. Zhou et al. reported a method to harvest intact cell sheets from standard culture dishes using a cell scraper, which made the use of cell-sheet technology more convenient and widely applicable [17]. Our group demonstrated that osteogenic cell sheets (OCSs) fabricated from BMSCs with dexamethasone (Dex) and ascorbic acid phosphate (AscP) can form bone tissue without the need for a scaffold after transplantation [18]. OCSs derived from different cell sources have been used in the production of vascularised tissue-engineering constructs [9,19], for the regeneration of periodontitis defects [20] and to enhance tendon–bone healing [21]. In a recent study from our group, we demonstrated that an OCS can enhance bone formation in a rat nonunion model [22]. Further, the percutaneous injection of an entire OCS was previously reported by our group, and the OCS, injected into subcutaneous sites on the dorsal surface of rats, formed the bone in the absence of a scaffold [23]. However, whether this minimally invasive approach encourages hard tissue reconstruction at bony sites and induces fracture repair has not yet been tested. To this end, the aim of the present study was to determine whether the percutaneous injection of entire OCSs enhances bone regeneration in order to demonstrate a novel method for the minimally invasive treatment of cases with compromised bone healing.
Materials and methods Study design Sixty male Fischer 344 inbred rats were divided into two groups: OCS and control groups (n = 30 each). A femoral critical fracture healing model was created in each rat, and the rats were sutured. After 6 h, two sheets of OCS in 1-ml phosphate-buffered saline (PBS) (Gibco, Invitrogen, Carlsbad, CA, USA) were injected into the osteotomy site in rats in the OCS group. Rats in the control group received an injection of PBS only. Femora were harvested from each group at 4, 8 and 12 weeks after the injection. Fracture healing was evaluated with radiological and histological analyses at 4, 8 and 12 weeks (n = 4). Micro-computed tomography (CT) and biomechanical analyses were also performed at 4, 8 and 12 weeks (n = 6). Ten additional rats were used to prepare sufficient quantities for injection. This research protocol was approved by the Institutional Animal Care and Use Committee of Nara Medical University, following all appropriate guidelines. Critical fracture healing model of the femur We modified a rat femur critical fracture healing model described previously [22]. Briefly, 12-week-old male Fischer 344 inbred rats (approximately 280 g) were anaesthetised with 2% isoflurane. The operative site was shaved and prepared with ethanol. A 5-cm lateral incision was made on the hind limb parallel to the femoral shaft, extending from the femoral condyle to the proximal part of the femur. The midshaft of the femur was exposed by dividing the vastus lateralis and the biceps femoris muscles, and a fracture was created by transverse osteotomy with an oscillating mini saw. The femoral canal was reamed with an 18-G needle (Terumo, Tokyo, Japan), and it was irrigated with 20 ml of sterile saline so that the bone marrow was completely ablated. The periosteum was removed as much as possible from the proximal to the distal ends of the femur, and the muscle attached to the periosteum was also removed. A K-wire (0.8 mm in diameter) was then inserted into the medullary canal in a retrograde fashion with the use of a motor-driven drill. The K-wire was positioned within the proximal part of the femur, and the distal end was then cut close to the articular surface of the knee. The wound was then irrigated with 10 ml of sterile saline, and it was closed with a 4/0 nylon suture. Unprotected weight bearing was allowed immediately after the operation. Postoperative pain was managed by the subcutaneous administration of buprenorphine hydrochloride. Postoperative antibiotics were administrated by intramuscular injection of penicillin prophylactically. The rats were fed a standard maintenance diet, and they were provided water ad libitum. BMSCs preparation from bone marrow aspirates The method for BMSCs preparation has been reported previously [18,22,23]. Briefly, bone marrow was obtained from the femoral shafts of 7-week-old male Fischer 344 inbred rats. Both ends of the femur were cut at the epiphysis and the bone marrow was aspirated from the marrow cavity using 10 ml of standard culture medium expelled from a syringe using a 21-G needle. Standard culture medium consisted of minimal essential medium (MEM) (Nacalai Tesque Inc., Kyoto, Japan) containing 15% fetal bovine serum (FBS) (Gibco, Invitrogen, Carlsbad, CA, USA) and 1% antibiotics (10,000 U/ml penicillin and 10,000 mg/ml streptomycin, Nacalai Tesque Inc.). Harvested cells were then transferred into two T-75 flasks (Falcon, BD, NJ, USA) containing 15 ml of standard culture medium. Cell culture was maintained in 95% humidified atmosphere with 5% CO2 at 37 8C. After reaching confluence,
T. Shimizu et al. / Injury, Int. J. Care Injured 46 (2015) 1457–1464
cultured cells were released from the culture substratum using trypsin/ethylenediamine tetra-acetic acid (EDTA) (Gibco). Preparation for OCS The method of OCS preparation has been previously reported [18,22,23]. Briefly, BMSCs were released using trypsin/EDTA, seeded at a cell density of 1 104 cells/cm2 in a 10-cm dish (100 mm 20 mm; Falcon, BD Biosciences, Franklin Lakes, NJ, USA) and cultured in a standard medium with 10-nM Dex (Sigma– Aldrich, St. Louis, MO, USA) and 82-mg/ml AscP (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 14 days. The cells were rinsed twice with PBS, and they were lifted as an OCS using a cell scraper. Total number of cells in an OCS was calculated based on the amount of DNA, which was determined with a DNA Quantification Kit (Primary Cell, Hokkaido, Japan) according to the instructions provided by the manufacturer. DNA analysis was performed with Hoechst 33258 Fluorometry. Briefly, the lifted OCS was digested with 2 ml of buffer solution, and it was lysed by 15-min sonication in an ice bath (n = 6). The samples (50 ml) of digestion and standard were also mixed with 1 ml of buffer solution and added with 50-ml working solution for 10 min at room temperature. Fluorescence of the sample mixture was analysed by a spectrophotometer (458 nm). Based on the linear relationship between fluorescence value and standard sample, a conversion factor was gained to calculate the cell number in an OCS. Suspended post-differentiated BMSCs were also collected from an OCS using trypsin/EDTA, and they were used to generate a standard. Based on the standardization, a linear relationship was obtained between the optical density and the suspended cell number (correlation R2 = 0.9621). The calculated cell number contained in an OCS in a 10-cm dish was averaged as 3.1 0.7 106 cells. Samples for injection Approximately 6 h after the first surgery, the rats were again sedated. Samples, prepared mentioned above, in 1-ml PBS were injected percutaneously into the fracture site of rats in each groups under the X-ray fluoroscopy using a 1-ml syringe (JMS Co., Ltd., Tokyo, Japan) to which a 16-G needle (Terumo, Tokyo, Japan) was attached. The injection was carefully applied anteriorly and posteriorly to the osteotomy site. This procedure was sophisticated with a contrast medium under the X-ray fluoroscopy beforehand in the pilot study, and we confirmed that samples were absolutely injected at the osteotomy site. Quantitative reverse transcriptase-polymerase chain reaction analysis for OCS Gene expression levels of alkaline phosphatase (ALP), osteocalcin (OC), runt-related transcription factor 2 (Runx2), SP7 (osterix), bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor-A (VEGF-A) were measured to assess the in vitro osteogenic and angiogenic potential of the OCS. Briefly, cells from the OCS and BMSCs were cultured in six-well plates for 14 days in osteogenic and standard media, respectively. RNA was harvested using an RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s protocol, and it was converted to complementary DNA (cDNA), as previously described [22]. The messenger RNA (mRNA) expression levels were assessed using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) (ABI PRISM 7700 Sequence Detection System, Applied Biosystems, Foster City, CA, USA), using the respective primers and probe set purchased from Applied Biosystems: ALP:
1459
Rn 00564931 m1, OC: Rn01455285 g1, Runx2: Rn01512298 m1, SP7: Rn02769744 s1, BMP-2: Rn00567818 m1, VEGF-A: Rn01511602 m1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH): Rn99999916 s1. Thermal cycling conditions were 10 min at 95 8C for the activation of the universal mixture AmpliTaq gold polymerase, followed by 40 cycles of 15 s at 95 8C for denaturing and 1 min at 60 8C for annealing and extension. Target mRNA levels were compared after correcting to GAPDH mRNA levels, which was used to adjust for differences in the efficiency of reverse transcription between the samples. Macroscopic and histological examination of OCS To explore how the process of injection into the defect site would alter the integrity of the cell sheet, we performed an in vitro test where OCSs were harvested by cell scraping and then injected into 10-cm dishes using the same needle and syringe used for the in vivo injections. The OCSs before and after in vitro injection were fixed in 10% neutral-buffered formalin solution (Wako Pure Chemical Industries) for 2 days, embedded in paraffin, cut parallel across the middle of the sheet and stained with hematoxylin and eosin (H&E) for light microscopic observation. Radiological evaluation Radiographs were taken at 4, 8 and 12 weeks after surgery for rats in all three groups. Animals were placed in a prone position with both limbs fully abducted and externally rotated under anaesthesia. Fracture union was determined by the presence of bridging callus on two cortices. Micro-CT and biomechanical evaluation Both treated and contralateral femurs (intact femur) were harvested from six rats in each group at 4, 8 and 12 weeks, and they were processed for micro-CT and biomechanical evaluation. Micro-CT was performed using a SMX-160CTS micro-CT (Shimadzu, Kyoto, Japan). Intramedullary fixation pins were removed prior to micro-CT analysis. After scanning, the femurs were subjected to standardised three-point bending tests performed using a universal testing machine (EZ graph, Shimadzu). The cross-head speed was 10 mm/min, with the maximum bending load-to-rupture determined as the ultimate load. The relative ratio (percentage maximum bending load) of the treated femur to the contralateral femur was calculated in each group. Histological evaluation For histology at 4, 8 and 12 weeks, the femurs were harvested from two rats in each group, and they were fixed in 4% paraformaldehyde in 0.1-M phosphate buffer (Wako Pure Chemical Industries) for 48 h. Subsequently, each femur was decalcified with 10% EDTA (Wako Pure Chemical Industries) for approximately 3 weeks, and it was embedded in paraffin. Paraffin sections (3 mm thick) were cut and stained with H&E and toluidine blue for histological evaluation. Statistical analysis All values are expressed as the mean standard deviation (SD). Mann–Whitney U-test was performed for comparisons between two experimental groups. Data analysis was conducted using the Statistical Package for Social Science (SPSS) software package (version 17.0; SPSS Inc., Chicago, IL, USA). A probability value p < 0.05 was considered to denote statistical significance.
1460
T. Shimizu et al. / Injury, Int. J. Care Injured 46 (2015) 1457–1464
Results Macroscopic and histological examination of OCS The macroscopic appearance of OCS after injection was quite similar to before harvesting, as determined using the in vitro injection assay. After injection, we observed that the OCS returned to their original shape within a few minutes (Fig. 1A). A histological evaluation of the OCS showed that the sheets folded over themselves, resulting in thick layered, cell-based structures (Fig. 1B). qRT-PCR expression for markers of osteogenesis in OCS and BMSCs In the in vitro analysis, the relative mRNA expression of ALP, OC, Runx2, SP7 and BMP-2 was significantly greater in the OCS group compared with that in the BMSC group. However, the relative mRNA expression of VEGF-A was lower in the OCS group compared with that in the BMSC group (Fig. 2). Radiological evaluation The OCS group displayed abundant callus formation around the fracture site at 4 weeks after OCS injection. By 8 weeks, the callus bridged both ends of the fractured bone, and the cortical gap on both sides of the defect site disappeared. This was later followed by callus consolidation by 12 weeks. None of the surrounding soft tissues showed isolated ossification from the cortex for rats treated with OCS at 12 weeks. By comparison, the control group showed a lack of vigorous callus formation around the fracture sites at 4 weeks. Although a small amount of callus formation was observed along the periosteum away from the fracture site at 8 weeks, this never extended to bridge the fracture site. By 12 weeks, the ends of the fractured bone had become rounded, and they were resorbed, giving the appearance of typical established nonunion (Fig. 3).
decrease in the thickness of the woven bone. The hard callus within the woven bone had been remodelled into the configuration of the host cortical and/or the trabecular bone. At 12 weeks, fracture healing was observed in the OCS group, but not in the control groups (Fig. 4). Histological evaluation In the OCS group, the osteotomy site was covered with newly formed bone at 4 weeks, and bridging ossification was observed at 8 weeks. By contrast, the control group demonstrated a large gap at the fracture site at 4 weeks, and by 8 weeks, the control group showed a fibrous tissue interposition between the ends of the fractured bone, which had become rounded and showed evidence of bony resorption. At 12 weeks, hard callus formation was observed in the OCS group, characterised by the formation of mineralized bone matrix in the absence of a cartilaginous template and a repopulation of the bone marrow in the canal. However, the control group showed large numbers of chondrocytes and fibrous granulation tissue at the fracture site, which is consistent with the characteristics of atrophic nonunion and non-bridging endochondral ossification at that time point (Fig. 5). Biomechanical evaluation The maximum bending load in the experimental femurs was determined across the course of the study. In cases where the experimental femurs did not rupture in the three-point bending tests, we determined the provisional maximum bending load to be that required to rupture the intact contralateral femurs (deflection of 1.2 mm). The percentage maximum bending load in the OCS group was significantly higher than those in the control group after 8 weeks (OCS: 69.4 16.8%, control: 7.9 7.8%, respectively, at 12 weeks). The percentage maximum bending load in the control groups was uniformly low throughout the experimental period (Fig. 6).
Micro-CT evaluation
Discussion
At 4 weeks, the micro-CT analysis demonstrated substantial new bone formation around the fracture site in the OCS group as compared with the control groups. This new bone formation continued to be observed at 8 weeks. By 12 weeks, the newly formed bone had united the two ends of the fracture, and it had started to undergo remodelling, as indicated by the progressive
The results of the present study show that the OCSs maintained their original structure after they were scraped and were successfully transplanted via a needle without causing too much disruption to the integrity of the sheet. Furthermore, injecting the entire OCSs into the osteotomy site of a rat femoral critical fracture healing model as a minimally invasive technique for bone repair
[(Fig._1)TD$IG]
Fig. 1. Macroscopic appearance and histological evaluation of osteogenic cell sheets (OCS). OCS was harvested by scraping, and it was injected into 10-cm dishes using a 1-ml syringe and a 16-G needle and cultured for 24 h. (A) The OCS returned to their pre-injection size within a few minutes. (B) Histology revealed that the OCS retained their cellsheet structure after injection.
[(Fig._2)TD$IG]
T. Shimizu et al. / Injury, Int. J. Care Injured 46 (2015) 1457–1464
1461
Fig. 2. Quantitative real-time PCR expression for markers of osteogenesis in osteogenic cell sheet (OCS) and BMSCs. Comparison of the in vitro gene expression changes after 14 days in culture under osteogenic or standard culture conditions, respectively. Values represent the mean SD for n = 5; *p < 0.05 versus BMSCs. All data were normalised to GAPDH transcript levels. Abbreviations: Alkaline phosphatase (ALP), osteocalcin (OC), bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF)-A, runtrelated transcription factor-2 (Runx2) and osterix (SP7).
has the potential to augment bone regeneration. The histological findings demonstrated a statistically significant increase in bone formation in the OCS group at 4 weeks and the formation of a bridging callus at the osteotomy site within the first 8 weeks. By
contrast, chondrocytes were present in the control group as late as 12 weeks, and the presence of fibrous granulation tissue between the bone fragments was consistent with the characteristics of atrophic nonunion at the fracture site. These results indicated the
[(Fig._3)TD$IG]
Fig. 3. Representative radiographs of fractured sites at 2, 4, 6, 8 and 12 weeks in OCS and control groups. The OCS group displayed abundant callus around the fracture site at 4 weeks. Between 8 and 12 weeks, the callus bridged both fractured ends, with a reduced frequency of gaps between the cortical bones. At 4 weeks, an absence of vigorous callus formation was noted in the control group around the fracture sites. Although small callus formation was seen along the periosteum away from the fracture site at 8 weeks, this failed to bridge the fracture site by 12 weeks.
[(Fig._4)TD$IG]
1462
T. Shimizu et al. / Injury, Int. J. Care Injured 46 (2015) 1457–1464
Fig. 4. Three-dimensional micro-CT evaluation of the OCS and control groups. The OCS group displayed newly formed bone around the fracture site at 4 weeks. At 8 weeks, the newly formed bone united both ends of the fracture, and the woven bone was remodeled into cortical and/or trabecular bone. At 12 weeks, fracture healing was observed in the OCS group, but not in the control groups. Scale bar: 3 cm.
utility of the percutaneous injection of entire scaffold-free OCSs as a minimally invasive surgical procedure for the treatment of cases with compromised bone healing. Osteoprogenitor cells play an important role in fracture healing, and they are contained within the periosteum [24] and bone marrow [25]. Furthermore, the circulation and vasculature [26,27], as well as the surrounding local tissues [28], offer an additional source of osteoprogenitor cells that has been implicated in bone formation and healing. Because clinical patients often exhibit delayed healing with compromised host bone tissue at fracture sites with reduced numbers of osteoprogenitor cells in their bone marrow [29], we used a rat critical bone healing model involving bone marrow ablation and the removal of femora periosteum, as previously reported by Kaspers et al. [30]. Peters et al. stated that this model appeared suitable for analysing the regenerative potential of transplanted cells in an osteogenic progenitor cellfree environment [31]. Thus, the effect of OCSs in this model system was isolated to generating the callus, and it did not induce the recruitment of endogenous osteoprogenitor cells.
Recently, Ma et al. demonstrated that the local injection of the fragments of multicellular aggregates produced from an OCS enhanced bone healing compared with that obtained in a cell-only control group in a rabbit model of delayed healing [32]. However, in their method, the OCS was cut into small, approximately 1-mmdiameter fragments to pass through the needle. This process may have disrupted the ECM within the OCS, as well as resulting in a loss in cell number. Moreover, it is technically demanding to cut a cell sheet into small fragments of approximately 1 mm in diameter with a scalpel, and it is easy to clog an 18-G syringe needle with the small fragments during aspiration. The ECM is responsible for transmitting a wealth of chemical and mechanical signals that mediate key aspects of cellular physiology, including adhesion, migration, proliferation, differentiation and death, in addition to providing support for cellular tissues and physical sites for cellular attachment [33]. Cell sheet technology preserves tight junctions between adjacent cells, which may be essential for maintaining apical–basal cell polarity [11], and it is useful for the preservation of deposited ECM during in vitro culture for bone tissue
[(Fig._5)TD$IG]
T. Shimizu et al. / Injury, Int. J. Care Injured 46 (2015) 1457–1464
1463
Fig. 5. Histological sections stained with fast green/safranin O for the OCS and control groups. The union process in the OCS group was quite different from the control groups. In the OCS group, the osteotomy site was covered with newly formed bone and bridging ossification at 4 and 8 weeks. By contrast, fibrous tissue was interposed between the ends of the fractured bone in the control groups. At 12 weeks, hard callus formation without cartilaginous template was observed in the OCS group, but the presence of chondrocytes and fibrous granulation tissue was observed at the fracture site in the control groups. Black scale bar: 500 mm; blue scale bar: 100 mm.
engineering applications [17]. In fact, the storage and release of various growth factors by the ECM produced by osteoprogenitor cells have been previously demonstrated [34]. Compared with previous methods [32], the percutaneous injection of entire OCSs
[(Fig._6)TD$IG]
Fig. 6. Biomechanical evaluation. The percentage (%) maximum bending load was increased in the OCS group throughout the course of the study. After 8 weeks, the percentage maximum bending load in the OCS group was significantly higher than those in the control groups. Values represent the mean SD for n = 6; *p < 0.05 versus control.
appears to maintain cell–cell interactions, and thus it offers distinct advantages for bone formation and regeneration. Therefore, the method described here improves and simplifies the previously reported methods, and it is an attractive, minimally invasive option that can be applied for the treatment of cases with compromised bone healing while maintaining the complex interplay of cells, growth factors and ECM. There are several limitations to this study. First, we used rat BMSCs. To translate this method into the clinic, we will need to assess the osteogenic potential of human OCSs for the treatment of cases with compromised bone healing. Second, we injected OCSs into the fracture site of the femur on the same day as when the osteotomy was created. However, in the clinical setting, the surgeon cannot use OCSs immediately during the initial operation because the OCS preparation requires several weeks of the BMSC expansion in culture. In practice, a surgeon would harvest the patient’s BMSCs during the initial operation for fracture treatment to create OCSs for use in later clinical cases. Future studies could elucidate the effects of the injection in a delayed union or in an established nonunion model using a variety of injection protocols, such as single versus multiple
1464
T. Shimizu et al. / Injury, Int. J. Care Injured 46 (2015) 1457–1464
injections. Third, other transplantation methods, besides the use of a 16-G needle as reported here, should be investigated. For example, Maeda et al. recently developed a device that allows the minimally invasive endoscopic transplantation of cell sheets fabricated in thermoresponsive dishes for the treatment of lung injury [35]. OCSs may also be applied for endoscopy-assisted bone reconstruction via small incisions. Conclusion Our results clearly demonstrate that the percutaneous injection of intact OCSs provides a minimally invasive technique to augment osteogenesis in cases with compromised bone healing. This minimally invasive technique therefore offers a promising approach for hard tissue reconstruction in the compromised bone healing situation. Not only for preventing nonunion or delayed union on high-risk patients, but our method could also be applied for several clinical applications, including augmentation of bone regeneration in segmental bone defects, osteonecrosis and distraction osteogenesis. Further experiments are necessary to validate the utility of this technique. Conflicts of interest statement There are no competing financial interests. Acknowledgements We thank Y. Kayukawa and N. Nitta (Doshisha University) for performing the micro-CT and the biomechanical evaluation, and F. Kunda, M. Yoshimura and M. Matsumura (Nara Medical University School of Medicine) for technical assistance. We also thank Y. Dohi, T. Ueha and A. Nakamura (Nara Medical University) for experimental advice. This work was supported by[1_TD$IF] [2_TD$IF]JSPS [3_TD$IF]KAKENHI Grant [4_TD$IF]Number 23592198. Grant support was also received from Japan Orthopaedics and Traumatology[5_TD$IF] Foundation, Inc. References [1] Pountos I, Georgouli T, George K, Giannoudis PV. Efficacy of minimally invasive techniques for enhancement of fracture healing: evidence today. Int Orthop 2010;34(1):3–12. [2] Schindeler A, McDonald MM, Bokko P, Little DG. Bone remodeling during fracture repair: the cellular picture. Semin Cell Dev Biol 2008;19(5):459–66. [3] Myeroff C, Archdeacon M. Autologous bone graft: donor sites and techniques. J Bone Joint Surg Am 2011;93(23):2227–36. [4] Hernigou P, Poignard A, Beaujean F, Rouard H. Percutaneous autologous bonemarrow grafting for nonunions. Influence of the number and concentration of progenitor cells. J Bone Joint Surg Am 2005;87(7):1430–7. [5] Muschler GF, Boehm C, Easley K. Aspiration to obtain osteoblast progenitor cells from human bone marrow: the influence of aspiration volume. J Bone Joint Surg Am 1997;79(11):1699–709. [6] Muschler GF, Nitto H, Boehm CA, Easley KA. Age- and gender-related changes in the cellularity of human bone marrow and the prevalence of osteoblastic progenitors. J Orthop Res 2001;19(1):117–25. [7] Xia Y, Mei F, Duan Y, Gao Y, Xiong Z, Zhang T, et al. Bone tissue engineering using bone marrow stromal cells and an injectable sodium alginate/gelatin scaffold. J Biomed Mater Res A 2012;100(4):1044–50. [8] Yamada Y, Nakamura S, Ito K, Kohgo T, Hibi H, Nagasaka T, et al. Injectable tissue-engineered bone using autogenous bone marrow-derived stromal cells for maxillary sinus augmentation: clinical application report from a 2–6-year follow-up. Tissue Eng Part A 2008;14(10):1699–707. [9] Zhao L, Tang M, Weir MD, Detamore MS, Xu HH. Osteogenic media and rhBMP2-induced differentiation of umbilical cord mesenchymal stem cells encapsulated in alginate microbeads and integrated in an injectable calcium phosphate–chitosan fibrous scaffold. Tissue Eng Part A 2011;17(7–8):969–79. [10] Anderson JM, Rodriguez A, Chang DT. Foreign body reaction to biomaterials. Semin Immunol 2008;20(2):86–100.
[11] Yang J, Yamato M, Kohno C, Nishimoto A, Sekine H, Fukai F, et al. Cell sheet engineering: recreating tissues without biodegradable scaffolds. Biomaterials 2005;26(33):6415–22. [12] Okano T, Yamada N, Sakai H, Sakurai Y. A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly (N-isopropylacrylamide). J Biomed Mater Res 1993;27(10):1243–51. [13] Nishida K, Yamato M, Hayashida Y, Watanabe K, Maeda N, Watanabe H, et al. Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface. Transplantation 2004;77(3):379–85. [14] Shimizu T, Yamato M, Isoi Y, Akutsu T, Setomaru T, Abe K, et al. Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces. Circ Res 2002;90(3):e40. [15] Kano K, Yamato M, Okano T. Ectopic transplantation of hepatocyte sheets fabricated with temperature-responsive culture dishes. Hepatol Res 2008;38(11):1140–7. [16] Kushida A, Yamato M, Isoi Y, Kikuchi A, Okano T. A noninvasive transfer system for polarized renal tubule epithelial cell sheets using temperature-responsive culture dishes. Eur Cell Mater 2005;10:23–30. [17] Zhou Y, Chen F, Ho ST, Woodruff MA, Lim TM, Hutmacher DW. Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts. Biomaterials 2007;28(5):814–24. [18] Akahane M, Nakamura A, Ohgushi H, Shigematsu H, Dohi Y, Takakura Y. Osteogenic matrix sheet-cell transplantation using osteoblastic cell sheet resulted in bone formation without scaffold at an ectopic site. J Tissue Eng Regen Med 2008;2(4):196–201. [19] Mendes LF, Pirraco RP, Szymczyk W, Frias AM, Santos TC, Reis RL, et al. Perivascular-like cells contribute to the stability of the vascular network of osteogenic tissue formed from cell sheet-based constructs. PLoS One 2012;7(7):e41051. [20] Uematsu K, Kawase T, Nagata M, Suzuki K, Okuda K, Yoshie H, et al. Tissue culture of human alveolar periosteal sheets using a stem-cell culture medium (MesenPRO-RSTM): in vitro expansion of CD146-positive cells and concomitant upregulation of osteogenic potential in vivo. Stem Cell Res 2013;10(1):1–19. [21] Chang CH, Chen CH, Liu HW, Whu SW, Chen SH, Tsai CL, et al. Bioengineered periosteal progenitor cell sheets to enhance tendon-bone healing in a bone tunnel. Biomed J 2012;35(6):473–80. [22] Nakamura A, Akahane M, Shigematsu H, Tadokoro M, Morita Y, Ohgushi H, et al. Cell sheet transplantation of cultured mesenchymal stem cells enhances bone formation in a rat nonunion model. Bone 2010;46(2):418–24. [23] Akahane M, Shigematsu H, Tadokoro M, Ueha T, Matsumoto T, Tohma Y, et al. Scaffold-free cell sheet injection results in bone formation. J Tissue Eng Regen Med 2010;4(5):404–11. [24] Hutmacher DW, Sittinger M. Periosteal cells in bone tissue engineering. Tissue Eng 2003;9(Suppl 1):S45–64. [25] Colnot C, Huang S, Helms J. Analyzing the cellular contribution of bone marrow to fracture healing using bone marrow transplantation in mice. Biochem Biophys Res Commun 2006;350(3):557–61. [26] Collett GD, Canfield AE. Angiogenesis and pericytes in the initiation of ectopic calcification. Circ Res 2005;96(9):930–8. [27] Eghbali-Fatourechi GZ, Lamsam J, Fraser D, Nagel D, Riggs BL, Khosla S. Circulating osteoblast-lineage cells in humans. N Engl J Med 2005;352(19): 1959–66. [28] Rumi MN, Deol GS, Singapuri KP, Pellegrini Jr VD. The origin of osteoprogenitor cells responsible for heterotopic ossification following hip surgery: an animal model in the rabbit. J Orthop Res 2005;23(1):34–40. [29] Sethe S, Scutt A, Stolzing A. Aging of mesenchymal stem cells. Ageing Res Rev 2008;5(1):91–116. [30] Kaspar K, Matziolis G, Strube P, Senturk U, Dormann S, Bail HJ, et al. A new animal model for boneatrophic nonunion: fixation by external fixator. J Orthop Res 2008;26(12):1649–55. [31] Peters A, Toben D, Lienau J, Schell H, Bail HJ, Matziolis G, et al. Locally applied osteogenic predifferentiated progenitor cells are more effective than undifferentiated mesenchymal stem cells in the treatment of delayed bone healing. Tissue Eng Part A 2009;15(10):2947–54. [32] Ma D, Zhong C, Yao H, Liu Y, Chen F, Li J, et al. Engineering injectable bone using bone marrow stromal cell aggregates. Stem Cells Dev 2011;20(6): 989–99. [33] Nelson CM, Bissell MJ. Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. Annu Rev Cell Dev Biol 2006;22:287–309. [34] Dallas SL, Rosser JL, Mundy GR, Bonewald LF. Proteolysis of latent transforming growth factor-beta (TGF-beta)-binding protein-1 by osteoclasts. A cellular mechanism for release of TGF-beta from bone matrix. J Biol Chem 2002;277(24): 21352–60. [35] Maeda M, Yamato M, Kanzaki M, Iseki H, Okano T. Thoracoscopic cell sheet transplantation with a novel device. J Tissue Eng Regen Med 2009;3(4):255–9.