Cell Biochem Biophys (2012) 63:171–181 DOI 10.1007/s12013-012-9354-1

ORIGINAL PAPER

Biological Characteristics and Effect of Human Umbilical Cord Mesenchymal Stem Cells (hUC-MSCs) Grafting with Blood Plasma on Bone Regeneration in Rats Zhiguo Qu • Libin Guo • Guojun Fang • Zhenghong Cui • Shengnan Guo • Ying Liu

Published online: 21 April 2012 Ó Springer Science+Business Media, LLC 2012

Abstract We evaluated the biological characteristics/ effect of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) grafting with blood plasma on bone regeneration in rat tibia nonunion. SD rats (142) were randomly divided into four groups: fracture group (positive control); nonunion group (negative control); hUC-MSCs grafting with blood plasma group; and hUC-MSCs grafting with saline group. Rats were administered tetracycline (30 mg/kg) and calcein blue (5 mg/kg) 8 days before killing. The animals were killed under deep anesthesia at 4 and 8 weeks post fracture for radiological evaluation and histological/immunohistological studies. The hUC-MSCs grafting with blood plasma group was similar to fracture group: the fracture line blurred in 4 weeks and disappeared in 8 weeks postoperatively. Histological/immunohistological studies showed that hUC-MSCs were of low immunogenicity which merged in rat bone tissue, differentiated into osteogenic lineages, and completed the healing of nonunion. After stem cell transplantation, regardless of whether plasma or saline was used, new multi-center bone formation was observed; fracture site density was better in stem cell grafting with blood plasma group. We, therefore, concluded that the biological characteristics of hUC-MSCs-treated nonunion were different from the standard fracture healing process, and the proliferative and localization capacity of hUC-MSCs might benefit from the use of blood plasma. Z. Qu  G. Fang  Z. Cui Department of Orthopaedic Surgery, Siping Central Hospital, Siping, Jilin, China L. Guo  S. Guo  Y. Liu (&) Department of Stem Cell Clinical Application Center, Siping Central Hospital, No. 89, Nanyingbin Road, Tiexi District, Siping 136000, Jilin, China e-mail: [email protected]

Keywords hUC-MSCs  Plasma  Stem cells Bone regeneration  Rat model

Introduction Ten percent of all fractures require further surgical procedures because of impaired healing [1]. Up to 17 % of nonunions were reported [2] after treatment of closed tibial shaft fractures. Impairment of fracture healing was linked to individual factors as well as demographic changes in society e.g., growth proportion of elderly people [3]. In orthopedic field, the use of autologous bone grafting remains the standard of care for clinical practice whereas it is limited in the availability of graft material and donor site morbidity [4]. Another approach to the treatment of nonunion fractures is to harness and expand the inherent osteogenic potential of bone marrow cells. It was shown that whole bone marrow combined with ceramic carriers could promote bone repair in animal models [5, 6]. A more recent study used carrier matrix to concentrate the marrow-derived osteoprogenitor cells and to also function as a scaffold for bone repair [7]. However, none of these methods used whole bone marrow to expand the osteoprogenitor cell pool and are, therefore, limited to the number of osteogenic donor cells that can be harvested from the patient. Patient-specific cell therapy, in many cases, requires large numbers of cells to replace the damaged or diseased tissue. Therefore, for clinical applications, a simple, safe, and reproducible method to effectively expand the osteogenic pool of cells is required. Bone marrow represents the most commonly used tissue source of adult mesenchymal stem cells (MSCs). Bone marrow mesenchymal stromal cells (BMSCs) have been used for cell-based therapies, including bone repair [8, 9]. MSCs are able to differentiate into various cell types

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including chondrocytes, osteocytes, adipocytes, myocytes, and neurons [10]. However, due to the limited number of BMSCs available for autogenous use and the possibility of donor site morbidity, there is a need to identify alternative MSC sources. Lately, the connective tissue (Wharton’s Jelly) of human umbilical cord (hUC-MSCs) was reported as a potential alternative tissue source of MSCs [11–13]. We previously reported transplantation of hUC-MSCs for treatment of nonunion [14], however, another study [15] reported expression of a set of factors related to osteogenesis and immune system regarding the use of hUC-MSCs [15]. Therefore, in the present study, we used hUC-MSCs transplantation for treatment of rat nonunion to assess the local effects of bone regeneration in rat tibial shaft nonunion as well as minimize the effects of systemic factors in this fracture. The primary aim of this study was to examine the biological characteristics of hUC-MSCs on the course of impaired bone healing in vivo. Herein, we report that after hUC-MSCs transplantation, with or without plasma, new multi-center bone growth formation was observed, whereas the fracture site density was better in stem cell grafting with blood plasma group. The biological characteristics of hUC-MSCs-treated nonunion were different from those of the standard fracture healing process.

Materials and Methods Umbilical Cord (UC) Harvesting Five human UC samples were collected following informed written consent from the mothers. The study protocol was approved by the institutional ethics committee and all experimental animal procedures were in accordance with the national/international guidelines for ethical conduct in the care and use of animals. Regarding each sample, UC sections of 8–10 cm were internally washed with phosphate buffered saline (PBS) containing 300 U/ml penicillin and 300 g/ml streptomycin (Gibco, NY, USA) and immediately immersed in Dulbecco’s modified Eagle’s medium—low glucose (DMEMLG, Gibco, NY, USA) supplemented with 10 % fetal bovine serum, 300 U/ml penicillin, and 300 g/ml streptomycin. All samples were processed within 12–15 h after collection. Isolation and Culture of Adherent Cells from UC UCs were filled with 0.1 % collagenase (Sigma-Aldrich, St. Louis, USA) in PBS and incubated at 37 °C for 20 min as described [16]. Each UC was washed with proliferation medium and the detached cells were harvested after gentle massage of the UC. Cells were centrifuged at 3009g for

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10 min, resuspended in proliferation medium, and seeded in 25-cm2 flasks at a density of 5 9 107 cells/ml. After incubation for 24 h, non-adherent cells were removed and culture medium was replaced every 3 days. Adherent cells were cultured until they reached 80–90 % confluence. Immunophenotyping To analyze cell-surface expression of the typical protein markers, adherent cells were incubated with the following anti-human primary antibodies (Becton–Dickinson Co., NJ, USA): CD31-phycoerythrin (PE) conjugated; CD45-fluorescein isothiocyanate (FITC) conjugated; CD90-R-PE conjugated; and HLA-DR-R-PE conjugated. Unconjugated markers were reacted with anti-mouse PE-conjugated secondary antibody (Guava Technologies, CA, USA). A total of 10,000 labeled cells were analyzed using a BD LSR TortessaÒ Cell Analyzer (San Jose, CA, USA) and the data obtained were analyzed using BD FACSDiva software 6.0 (San Jose, CA, USA). Experimental Non-Union Model The study protocol was approved by the institutional animal care and use committee and conformed to the recommended guidelines. In this study, 142 Sprague-Dawley (SD) rats were used. The mean age of the rats was approximately 7 (range: 6–8) weeks and mean body weight was approximately 250 (range: 226–268) grams. All surgical procedures were performed under anesthesia and normal sterile conditions. Anesthesia was induced with 4 % halothane inhalation, followed by ketamine hydrochloride (80 mg/kg) administered intraperitoneally (i.p.). The rats were randomly and equally divided into following four groups (with equal weight distributions): (1) fracture group as a positive control; (2) non-union group as a negative control; (3) hUC-MSCs grafting with saline group; and (4) hUC-MSCs grafting with blood plasma group. Rats were killed under deep anesthesia at 4 and 8 weeks after fracture. A lateral parapatellar knee incision was made on the right limb to expose the distal femoral condyle. A 1.25 mm diameter k-wire was inserted from the trochlear groove into the tibial canal in a retrograde fashion using a motor-driven drill. The wire was advanced through the greater trochanter and out of the skin until its distal end was positioned deep into the articular surface of the knee. A 5-mm incision in the skin was made around the k-wire and the wire was then cut close to the proximal femur. After irrigation, the wounds were closed with 5-0 nylon suture. Then transverse mid-diaphyseal tibial fracture was induced with microlandscape. Following this procedure, half (64) of the rats received additional surgery to create the nonunion in the fractured

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shaft. In order to produce the nonunion, the fracture site was minimally exposed through a lateral approach. The periosteum was then cauterized (Loop tip surgical cautery, Abco Dealer Inc., TN, USA) circumferentially for a distance of 2 mm on each side of the fracture. The muscle was protected to preserve all soft tissue except the periosteum around the fracture site. We were careful to cauterize the periosteum only once to prevent excess thermal necrosis of bone. The wound was irrigated with 10 ml of sterile saline and muscle and the skin were closed in layers with 5-0 nylon sutures. Post-operative pain was managed by administration of subcutaneous injection of buprenorphine hydrochloride. The rats were fed a standard maintenance diet and provided water ad libitum. Unprotected weight bearing was allowed immediately post-operatively. The left non-fractured femur served as a control. Equal numbers of animals were assigned to each group and were maintained for the intervals of 4 and 8 weeks. Eight specimens from each time point were randomly selected for histological examination. If the fracture produced was not a stable transverse fracture or if evidence of deep infection developed then the animal was exclude from the study and replaced with another animal. Thus, 10 rats with comminuted fracture and four rats with infection were replaced during the experiment. Animals were regularly monitored radiographically. Mediolateral and anteroposterior radiographs were taken postoperatively, and also at 4 and 8 weeks following surgery. For histomorphometric evaluations as described previously [17], tibia samples were harvested after injections of tetracycline (30 mg/kg) and calcein blue (5 mg/kg) at 8 days before death. After removing soft tissues, samples were fixed in 70 % ethanol and embedded in polymethylmethacrylate. A conventional bone histological evaluation was performed.

sliced into 3-lm thick sections following the standard method. The slides were rinsed twice in PBS, followed by a rinse in PBS containing 0.25 % triton X-100 (PBS-TX). The sections were incubated overnight in a dark humid chamber at room temperature with rabbit anti-human ANA (US Biological C7150-13B) or rabbit anti-BMP-2 (US Biological C7150-13B) 1:200 diluted in PBX-TX containing 1 % bovine serum albumin. After several washes in PBS, the sections were incubated for 1 h in a dark humid chamber at room temperature with goat anti-rabbit IgG conjugated to Alexa488 (Molecular Probes/Invitrogen) or anti-rabbit IgG conjugated to Dylight594 (Molecular Probes/Invitrogen) 1:200 diluted in PBS containing 1 % bovine serum albumin. The sections were rinsed several times in PBS, mounted on cover-slips in FluoroSave mounting medium and visualized under a Nikon Eclipse 800 fluorescent microscope (Nikon Instruments, NY, USA). Stained cells were counted in each slice by three blinded independent observers to assess the proliferative, localization, and differentiation potential of hUC-MSCs in the blood plasma and saline groups.

Transplantation of hUC-MSCs

At the end of the maintenance intervals, 32 rats allocated for histological evaluation were euthanized with an excess of carbon dioxide gas. After removing soft tissues, right femurs were harvested and fixed in gradient ethanol for dehydration for 14 days and then embedded in polymethylmethacrylate. For histological examination, 50-lm thick sections were prepared and stained by Van Gieson staining method. The histological examination was performed to confirm that the standard fracture closure model represented normal stages of fracture healing, whereas the nonunion model represented an established nonunion.

The rats were positioned supine decubitus on the operation table and the left thigh was disinfected with iodophor. Then, hUC-MSCs reconstituted blood plasma was slowly injected (1 ml) using epidural needle into fracture site through the skin in front of the thigh, the needle was withdrawn and the final 0.3 ml of stem cells with blood plasma was injected around the fracture site and sterilized dressing was wrapped around the puncture site. The animals were maintained in supine decubitus recumbency for another 30 min and then shifted to individual cages. Antibiotics were administered postoperatively to prevent infection.

Radiological Evaluation Radiographs were obtained for all rats following creation of the fracture and at two time points before killing. For this, anesthesia was induced and the animals were laid in prone position with both limbs fully abducted. Fracture union was determined by the presence of bridging callus on two cortices. Radiographs of each animal were assessed by three blinded independent observers to assess whether or not the fractures were united. Histological Evaluation

Statistical Analysis Immunofluorescence Tibias were embedded in paraffin wax after decalcification in buffered EDTA (14.5 %; pH 7.2) for 2 weeks and were

The data obtained were expressed as means ± SD values and compared between groups using one-way ANOVA. All P values \0.05 were considered statistically significant.

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Results Isolation and Culture of Adherent Cells from UC All UC samples generated primary adherent cultures with cells displaying an MSC-like phenotype. After 4 days in culture, these cells grew in colonies, reaching confluence after 10–14 days. Most of the cells were spindle-shaped, resembling fibroblasts. After the second passage, adherent cells were constituted by homogeneous cell layers with an MSC-like phenotype (Fig. 1a, b). The number of MSC from UC decreased slightly after freezing and thawing, and the remaining viable cells were successfully expanded on consecutive days (data not shown). Immunophenotypic Analysis All adherent cells derived from UC did not express hematopoietic lineage markers (CD45) and endothelial markers (CD31), HLA-DR (HLA-class II) as assessed by flow cytometry (Fig. 2a–c). In addition, the majority of cells expressed high levels of CD90 adhesion marker (Fig. 2d); and in comparison with the fibroblast control, no obvious difference in the expression of these surface antigens could be observed (data not shown).

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obvious external callus. Radiographic assessment of stem cells grafting with blood plasma group was similar to that of fracture group. In case of stem cells grafting with saline group, the fracture line was still visible (Fig. 3d). Apart from fracture group, a part of continuous bridging callus could be seen in stem cell grafting with blood plasma group and stem cells grafting with saline group (Fig. 3c, d). At 8 weeks after fracture, the nonunions did not have much callus formation around the fracture sites. As though some callus did form along the periosteum away from the fracture site, it never expanded to bridge the fracture site (Fig. 3e). The ends of the fractured bone became round, hardened, and formed pseudarthrosis. On the other hand, the fracture line disappeared in fracture group (Fig. 3f) and there was an obvious external callus formation. Stem cell grafting with blood plasma group was similar to fracture group in which most of the callus bridged between both ends of the fractured bones and the cortical gaps disappeared (Fig. 3g, h). Fracture line blurred in stem cells grafting with saline group and much of the callus formation occurred around the fracture sites (Fig. 3i). Histological Analysis

Radiographs taken just after surgery showed transverse mid-diaphyseal femoral fracture in nonunion model (Fig. 3a). In case of nonunion, the fracture line remains visible, fractured bone ends were absorbed and atrophied (Fig. 3b). At 4 weeks postoperatively, the fracture line blurred in fracture group (Fig. 3c) and there was no

At 4 weeks after induction of fracture, in nonunion group, the gap between the calluses was wider. The fracture group rats displayed intramembranous ossification in the periosteal tissue and endochondral ossification at the fracture site (Fig. 4b). A thick callus formed that consisted of chondrocytes and newly-formed trabecular bone, and the two calluses in each side of the fracture almost united. The gap between endochondrocytes and endochondral ossification in stem cell grafting with blood plasma group were similar to those observed in fracture group (Fig. 4c) but there was no bone

Fig. 1 Isolation and culture of adherent cells from umbilical cord (UC). a All UC samples generated primary adherent cultures, with cells displaying mesenchymal stem cell (MSC)-like phenotype. After

4 days in culture, these cells developed colonies. b Cell cultures showed confluence after 10–14 days and most of the cells were spindle-shaped

Radiographic Analysis

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Fig. 2 Immunophenotypic analysis. a Immunostaining for CD45 shows that all adherent cells derived from UC do not express CD45. b Immunostaining for CD31 shows that all adherent cells derived from UC do not express CD31. c Immunostaining for HLA-DR shows that all adherent cells derived from UC do not express HLA-DR. d The majority of the cells express high levels of CD90

formation on the site of periosteal cauterization. In addition, in stem cells grafting with saline group, the gap between the calluses was smaller than that of nonunion group and the callus formed was thin (Fig. 4d). In contrast, a large gap persisted between the surfaces of woven bone in the nonunions (Fig. 4e). At 8 weeks after fracture induction, the callus in fracture group had united and chondrogenic areas almost disappeared (Fig. 4f). The fractured bone was covered with newly-formed trabecular bone and achieved bony union. In stem cell grafting with blood plasma group, similar to fracture group (Fig. 4g), the fractured bone was covered with newly-formed trabecular bone and achieved bony union but bone marrow cavity was thinner. Notably, at 8 weeks, the united bone in fracture group had remodeled with a progressive decrease in the thickness of the woven bone (Fig. 4f). The fracture gap at the interface of the original cortical bone was indistinguishable. However, in nonunion model at 8 weeks, the fibrous tissue surrounded the fracture site and resorption of the end of the cortical bone had started (Fig. 4e). This was consistent with the histological presentation of atrophic nonunions. In stem cells grafting with saline group, the fractured bone was covered with newly-formed trabecular bone but it had not as yet achieved bony union (Fig. 4h).

We also observed new bone formation after injections of tetracycline (30 mg/kg) and calcein blue (30 mg/kg) at 8 days before killing. In nonunion group, we observed new bone formation at both 4 and 8 weeks after fracture (Fig. 5a, e) but the new bone could not form a continuous curve. In fracture group (Fig. 5b, f), the healing process of fracture was in accordance with the standard healing process as it displayed intramembranous ossification in the periosteal tissue and endochondral ossification at the fracture site. After stem cells transplantation, regardless of whether plasma or saline was used, a new multi-center bone formation occurred; however, the density in the fracture site was better in stem cell grafting with blood plasma group (Fig. 5c, d, g, h). Immunohistological Findings To further study the biological characteristics of hUCMSCs, we examined anti-human antinuclear antibody (ANA)-labeled hUC-MSCs in calluses of osteotomized rat tibiae in stem cell transplantation groups at 8 weeks after fracture (Fig. 6). A thicker callus formed in stem cell grafting with blood plasma group. Labeled hUC-MSCs

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Fig. 3 Radiographic analysis. a Radiographs taken immediately after surgery show transverse mid-diaphyseal femoral fracture in nonunion model. b At 4 weeks postoperatively, the fracture line is still visible, and fractured bone ends show absorption and atrophy in nonunion groups. c At 4 weeks postoperatively, the fracture line blurred in fracture group; there is no obvious external callus, but a part of continuous bridging callus can be seen. d At 4 weeks postoperatively, stem cell grafting with blood plasma group was similar to fracture group. The fracture line blurred, there is no obvious external callus and a part of continuous bridging callus can be seen. e At 4 weeks postoperatively, in stem cells grafting with saline group, the fracture line is still visible, however, a part of continuous bridging callus can be seen. f At 8 weeks post fracture, the nonunions did not have much

callus formation around the fracture sites. Some callus did form along the periosteum away from the fracture site but it never expended to bridge the fracture site. The ends of the fractured bone became rounded and hardened to form pseudarthrosis. g At 8 weeks post fracture, the fracture line disappeared in fracture group, there was obvious external callus and the cortical gaps disappeared. h At 8 weeks post fracture, stem cell grafting with blood plasma group was similar to fracture group—most of the callus bridged between both ends of the fractured bones and the cortical gaps disappeared. i At 8 weeks post fracture, the fracture line blurred in stem cells grafting with saline group and much of the callus formation was around the fracture sites

were identified by green fluorescence (Figs. 7a, 8a). In the calluses of osteotomized rat tibiae, large numbers of labeled cells were observed. Moreover, the numbers of labeled cells in these calluses were significantly higher (P \ 0.05) than those of hUC-MSC grafting with saline group (Figs. 7a, 8a). Anti-human ANA-labeled hUC-MSCs expressed bone morphogenetic protein (BMP)-2; visible as red fluorescence in yellow fluorescence in calluses of osteotomized rat tibiae (Figs. 7d, 8d) whereas green fluorescence indicated that some colonized hUC-MSCs did not express BMP-2. Besides, as shown in Figs. 7d and 8d, we

observed small, solitary red fluorescence which indicated that rat cells stimulated by nonunion could also begin to proliferate.

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Discussion In order to evaluate an applicable in vivo model for local therapeutic targeting, we investigated the impaired course of healing of a rat tibial nonunion with intramedullary stabilization and/or hUC-MSCs as compared to a tibial

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Fig. 4 Histological analysis (Van Gieson staining). a At 4 weeks post fracture, in the nonunion group, the gap between the calluses was wider. b The fracture model rats display intramembranous ossification in the periosteal tissue and endochondral ossification at the fracture site. They formed a thick callus consisting of chondrocytes and newly-formed trabecular bone, and the two calluses on each side of the fracture were almost united. c The gap between endochondrocytes and endochondral ossification in stem cells grafting with blood plasma group were similar to those of fracture group but there was no bone formation on the site of periosteal cauterization. d In addition, in stem cells grafting with saline group, the gap between the calluses was smaller than in nonunion group, and the callus formed was thin. e At 8 weeks post fracture, a large gap persisted between the surfaces

of woven bone in the nonunions, and the end of the cortical bone was resorbed. f The callus in the fracture group had united and the chondrogenic areas almost disappeared. The fractured bone was covered with newly-formed trabecular bone, achieved bony union, and remodeled with progressive decrease in the thickness of the woven bone. The fracture gap at the interface of the original cortical bone was indistinguishable. g In stem cells grafting with blood plasma group, similar to fracture group, the fractured bone was covered with newly-formed trabecular bone, achieved bony union, but bone marrow cavity was thinner. h In stem cells grafting with saline group, the fractured bone was covered with newly-formed trabecular bone but it did not achieve bony union just yet

osteotomy model with regular healing. The identification of the types of structural and biological variations regarding fracture healing in animal models, as used in this study, would be useful to assess the expected variation for human bone healing. The radio-histological screening used in our study showed a clear course of impaired bone healing. At 4 weeks after surgery, the fracture line blurred in fracture group and there was no remarkable external callus formation. The hUC-MSC grafting with blood plasma group compared with fracture group and the similar findings were observed in two groups. At 8 weeks after surgery, the fracture line disappeared in fracture group and a remarkable external callus formation was observed. Again, the hUC-MSC grafting with blood plasma group compared

with the fracture group, as well as most of the callus bridged between both ends of the fractured bones and the cortical gaps disappeared. There were insignificant differences observed between two groups. At 8 weeks after fracture, the nonunions did not have much callus formation around the fracture sites. Some callus did form along the periosteum away from the fracture site but it did not expand to bridge the fracture site. The ends of the fractured bone became rounded and were resorbed. Due to cauterization on each side of the fracture, the radiographic appearance of nonunions was atrophic as reported previously [18]. Compared with hUC-MSC grafting with blood plasma group, a delayed healing after tibial osteotomy was observed in hUC-MSC grafting with saline group at both 4

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Fig. 5 New bone formation analysis. a At 4 weeks post fracture, new bone formation in the nonunion group did not form a continuous curve. b In fracture group, the fracture healing was in accordance with the standard healing process. It displayed intramembranous ossification in the periosteal tissue and endochondral ossification at the fracture site. c New bone formation in stem cells grafting with blood plasma group is shown at 4 weeks after fracture. d New bone formation in stem cells grafting with saline group is shown at 4 weeks

after fracture. e New bone formation in nonunion group is shown at 8 weeks. f At 8 weeks post fracture, the fracture line disappeared in fracture group. g At 8 weeks post fracture, stem cell grafting with blood plasma group was similar to fracture group—most of the new callus bridged between both ends of the fractured bones and new multi-center dense bone growth was observed in the fracture site. h At 8 weeks post fracture, new bone formation in stem cells grafting with saline group is shown

Fig. 6 Immunohistological examination of callus in stem cells transplantation groups. Anti-human antinuclear antibody (ANA)labeled hUC-MSCs stained with 40 -6-diamidino-2-phenylindole (DAPI), a fluorescent nuclear stain, are shown to express bone

morphogenetic protein (BMP)-2 (purple fluorescence) in callus of osteotomized rat tibiae. a Callus is shown in stem cell grafting with saline group (green/red fluorescence). b Callus is shown in stem cell grafting with blood plasma group (green/red fluorescence)

and 8 weeks. Histological investigations, at 4 and 8 weeks postoperatively, also revealed differences between two groups. The histomorphometric analyses (Fig. 4) showed

that the amounts of reparative bone tissue and differentiated cartilaginous tissue were retarded in the region of the osteotomy defect in hUC-MSC grafting with saline group

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Fig. 7 Immunohistological evaluation in stem cell grafting with blood plasma group. a Anti-human ANA-labeled hUC-MSCs are shown in callus of osteotomized rat tibiae. b BMP-2 expression is shown by red fluorescence in callus of osteotomized rat tibiae. c DAPI expression is shown by blue fluorescence in callus of osteotomized rat tibiae. d Antihuman ANA-labeled hUCMSCs are shown to express BMP-2 (yellow fluorescence) in callus of osteotomized rat tibiae while the non-expressing cells are identified by green fluorescence. e Anti-human ANA-labeled hUC-MSCs stained with DAPI are shown to express BMP-2 (purple fluorescence) in callus of osteotomized rat tibiae

as compared to hUC-MSC grafting with blood plasma group. From these results, and also from our previously published data [16], we gather that the blood plasma could improve the proliferative and localization capacity of hUCMSCs. To further assess the biological characteristics of hUCMSCs, we examined anti-human ANA-labeled hUC-MSCs in calluses of osteotomized rat tibiae at 8 weeks after fracture. We observed that in stem cell transplantation groups, a thicker callus formed in stem cell grafting with blood plasma group. In the nonunions, the appearance of red fluorescence

spots indicated that that rat cells could be stimulated by nonunion to proliferate and express BMP-2. In the nonunions, although a new bone formation occurred, no course of healing could be observed. The bone cells near the fracture site as well as autologous stem cells/MSCs had mobilized to the fracture site and completely transformed into the boneforming cells. In this rat model study, the labeled hUC-MSCs were identified by their peculiar green fluorescence and we found large numbers of the labeled cells in the calluses of osteotomized rat tibiae. Besides, the numbers of labeled cells in calluses of hUC-MSC grafting with blood plasma group

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Fig. 8 Immunohistological evaluation in stem cell grafting with saline group. a Anti-human ANA-labeled hUC-MSCs are shown in callus of osteotomized rat tibiae. b BMP-2 expression is shown by red fluorescence in callus of osteotomized rat tibiae. c DAPI expression is shown by blue fluorescence in callus of osteotomized rat tibiae. d Antihuman ANA-labeled hUCMSCs are shown to express BMP-2 (yellow fluorescence) in callus of osteotomized rat tibiae while the non-expressing cells are identified by green fluorescence. e Anti-human ANA-labeled hUC-MSCs stained with DAPI are shown to express BMP-2 (purple fluorescence) in callus of osteotomized rat tibiae

were significantly higher than those of hUC-MSC grafting with saline group. Anti-human ANA-labeled hUC-MSCs observed in the calluses of osteotomized rat tibiae expressed BMP-2 (yellow fluorescence). In agreement with the previous studies [19–21], it also suggests that the hUC-MSCs used were of low immunogenicity which merged in rat bone tissue, differentiated into bone cells, and completed the healing of nonunion. Side by side, the appearance of solitary green fluorescence indicated that some hUC-MSCs’ colonies in rat calluses did not express BMP-2 which may be explained as follows: (1) in complete bone healing process of nonunions, only a part of the transplanted hUC-MSCs acted as the stem/

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progenitor cells; and (2) a part of the transplanted hUCMSCs failed to convert to bone cells in the body environment. Arguably, the first speculation appears to be more plausible. As shown in Figs. 6, 7 and 8, we did not observe solitary red fluorescence in woven bone cavity. We suggest that rat autologous cells migrated to the fracture site and completely transformed into bone-forming cells following the standard healing process of fracture i.e., with intramembranous ossification in the periosteal tissue and endochondral ossification at the fracture site. After stem cells transplantation, regardless of whether plasma or saline was used, new multi-center bone growth occurred. In case of

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hUC-MSCs grafting with saline group, we suggest that the transplanted hUC-MSCs might secrete some factors to favor the colonization of hUC-MSCs at the fracture site; and these factors might also contribute to the proliferation of bone cells in rats. In regard to hUC-MSCs grafting with blood plasma group, we gather that the proliferative and localization capacity of hUC-MSCs might benefit from the blood plasma constituents. In summary, this rat model study demonstrates that hUC-MSCs grafting with blood plasma has the capacity to proliferate and differentiate into the osteogenic lineages involved in standard process of bone healing. Thus, hUCMSCs can be used as an effective therapeutic intervention. Besides, local bone regeneration can also benefit from blood plasma constituents which may have important clinical implications.

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Acknowledgments We thank all the cord blood donors as well as hospital support staff for their cooperation. 15.

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