Vitreous cryopreservation of tissue engineered bone composed of bone marrow mesenchymal stem cells and partially demineralized bone matrix

Cryobiology 59 (2009) 180–187

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Vitreous cryopreservation of tissue engineered bone composed of bone marrow mesenchymal stem cells and partially demineralized bone matrix q Hongyu Yin a, Lei Cui b,c, Guangpeng Liu b, Lian Cen b, Yilin Cao a,b,c,* a

Plastic Surgery Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100041, China Shanghai Tissue Engineering Research and Development Center, Shanghai 200235, China c Department of Plastic and Reconstructive Surgery, Shanghai 9th People’s Hospital, Shanghai 200011, China b

a r t i c l e

i n f o

Article history: Received 11 February 2009 Accepted 29 June 2009 Available online 1 July 2009 Keywords: Vitrification Tissue engineering Bone marrow mesenchymal stem cells

a b s t r a c t Cryopreservation of tissue engineered products by maintaining their structure and function is a prerequisite for large-scale clinical applications. In this study, we examined the feasibility of cryopreservation of tissue engineered bone (TEB) composed of osteo-induced canine bone marrow mesenchymal stem cells (cBMSCs) and partially demineralized bone matrix (pDBM) scaffold by vitrification. A novel vitreous solution named as VS442 containing 40% dimethyl-sulfoxide (DMSO), 40% EuroCollins (EC) solution and 20% basic culture medium (BCM) was developed. After being cultured in vitro for 8 days, cell/scaffold complex in VS442 was subjected to vitreous preservation for 7 days and 3 months, respectively. Cell viability, proliferation and osteogenic differentiation of cBMSCs in TEB after vitreous cryopreservation were examined with parallel comparisons being made with those cryopreserved in VS55 vitreous solution. Compared with that cryopreserved in VS55, cell viability and subsequent proliferative ability of TEB in VS442 after being rewarmed were significantly higher as detected by live/dead staining and DNA assay. The level of alkaline phosphatase (ALP) expression and osteocalcin (OCN) deposition in VS442 preserved TEB was also higher than those in the VS55 group since 3 days post-rewarm. Both cell viability and osteogenic capability of the VS55 group were found to be declined to a negligible level within 15 days post-rewarm. Furthermore, it was observed that extending the preservation of TEB in VS442 to 3 months did not render any significant effect on its survival and osteogenic potential. Thus, the newly developed VS442 vitreous solution was demonstrated to be more efficient in maintaining cellular viability and osteogenic function for vitreous cryopreservation of TEB over VS55. Ó 2009 Published by Elsevier Inc.

Introduction Treatment of large bone defects caused by osseous infection, fracture nonunion, severe trauma, resection of tumors, and congenital malformations represents a challenge to orthopedic surgeons. To overcome the problems associated with existing biological grafts, such as auto- and allograft, bone tissue engineering has emerged as an alternative approach towards restoration of bone defects [24]. The application of tissue engineering approach to bone regeneration is currently described as a stepwise process: isolation of a small quantity of seed cells for expansion, preparation of scaffold, seeding the cells on the scaffold to form a cell/scaffold complex and implantation of the complex to fill bone defect [12]. Owing

q Statement of funding: Beijing Science and Technology Plan (Grant No.: H060920051230). * Corresponding author. Address: Plastic Surgery Hospital, Chinese Academy of Medical Sciences& Peking Union Medical College, Beijing 100041, China. E-mail address: [email protected] (Y. Cao).

0011-2240/$ - see front matter Ó 2009 Published by Elsevier Inc. doi:10.1016/j.cryobiol.2009.06.011

to their promising osteogenic differentiation potential both in vitro and in vivo, the multipotent bone marrow mesenchymal stem cells (BMSCs) combined with suitable scaffolds have been extensively used in repairing bone defects in either animal models [13] or human bodies [20]. Scaffolds, both from naturally derived [1] and artificial synthetic biomaterials [32], play a crucial role in supporting the proliferation and differentiation of seed cells via offering a 3-dimensional (3D) environment. As one of the classical naturally derived materials with osteoinductive properties, partially demineralized bone matrix (pDBM) exhibited good biocompatibility with BMSCs in vitro [5]. In previous studies [21], we demonstrated that tissue engineered bone (TEB) could be formed by transplanting osteogenic induced autologous BMSCs loaded on pDBM in cranial bone defect in a canine model. Thus, osteogenic differentiated BMSCs combined with pDBM have been proven to be a promising product to regenerate bone tissue for repairing purposes. However, from the practical point of view, preparation of TEB by the stepwise process mentioned above is time consuming, while clinical requirements especially as ‘‘off-the-shelf” products can not

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be meet. To circumvent this major obstacle in commercializing TEB, cryopreservation of the cell/scaffold complex for future clinical applications could be adopted [14]. In a previous study [19], we demonstrated that cryopreserved human BMSCs maintained their growth and osteogenic differentiation in vitro. More importantly, no effect on the osteogenic regeneration ability was observed when those BMSCs were transplanted with pDBM scaffold in vivo. However, the crucial question of whether BMSCs already seeded on 3D scaffold could resume their osteogenic potential after being cryopreserved remains unanswered. In addition, the damaging effect from the formation of ice crystals on cells and tissues during freezing in conventional cryopreservation process is well documented [11]. Vitrification, defined as a glass-like solidification, completely avoids the formation of ice crystals during cooling and warming, and is demonstrated to have great superiority in preserving cells [16] or organs [31] to those conventional freezing methods. It was shown that viability of cells in TEB after rewarming from vitreous preservation was three times that from traditional cryopreservation [17]. More recently, Bhakta et al. [3] reported that cryopreservation of constructs composed of BMSCs with alginate-fibrin beads via vitrification had no significant impact on osteogenic differentiation of the BMSCs as evidenced by calcium deposition. Choosing a suitable cryoprotective agent is a prerequisite for succeeding in vitreous preserving cell/ biomaterial constructs [14]. However, the commonly used cryoprotective solutions for vitrification, such as VS55, are generally toxic to cells in relatively high concentrations [17]. When VS55 was used to vitrify suspended osteoblast cells, it was found that only 83% of cell viability could be achieved after rewarming [18]. Thus, it is of great importance to find a superior vitreous solution to minimize the toxic side-effects but retain osteogenic capability of cell/scaffold constructs after vitreous preservation. In the present study, we investigated the feasibility of vitreous preservation of osteo-induced canine bone marrow mesenchymal stem cells (cBMSCs)/pDBM complex in two kinds of cryoprotective solutions. Growth, proliferation and osteogenic differentiation of cBMSCs in the constructs cryopreserved for 7 days and 3 months were examined and compared, respectively.

Materials and methods

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and subcultured as passage 1. cBMSCs of passage 2 (P2) were used for the following experiments. After cell plating, the in vitro osteogenic differentiation of cBMSCs at P2 was revealed by alkaline phosphatase (ALP) staining using the BM-purple method, ECM mineralization by Alizarin Red staining, and osteocalcin (OCN), osteopontin (OPN) and bone sialoprotein (BSP) staining by immunocytochemistry at days 14 as previously described [26]. Preparation of cBMSCs/pDBM constructs pDBM was prepared using porcine trabecular bone in a process described previously [5]. Briefly, cancellous bone was harvest from femur head of Shanghai white swine (from the animal holding center at Shanghai JiaoTong University School of Medicine). The samples were cleaned to remove the residual tissue, sectioned into discs (5 mm in diameter and 1 mm in thickness) (Fig. 1), decellularized by 1% Triton X-100 for 48 h, degreased with methane/ methyl alcohol, partially demineralized in 0.6 N HCl for 5 min, and finally rinsed and immersed in distilled water overnight at 4 °C. The scaffolds were then air dried for 24 h and sterilized by Ethylene Oxide before use. The pDBM discs had a volumetric porosity of 80.05 ± 3.92% and mean pore diameter of 330.5 ± 33.6 lm as measured by micro-CT. Calcium content of the scaffolds before and after demineralization was measured as reported previously [22]. In this study, the residual calcium content of pDBM was 16 ± 1% after demineralization, and 23 ± 2% before demineralization, so the demineralization extent was 29.5%. Osteogenic-induced cBMSCs were detached and resuspended at a density of 50  106 cells/mL. Twenty microliters of the cell suspension was slowly added onto the scaffolds by instillation and incubated for 4 h at 37 °C to allow cell attachment. Then the scaffolds were transferred into 24-well plates, and subcultured with 1 mL of OM. The medium was changed every 2–3 days after cell seeding. Proliferation and osteogenic differentiation of cBMSCs on scaffolds To observe the attachment and growth of seeded cells on the pDBM scaffolds, at days 1 and 8 after seeding, the TEB was viewed under a confocal laser microscope (CLM, Zeiss LSM Axiovert, Carl

Isolation, cultivation and osteogenic differentiation of cBMSCs A total of six adult Beagle canines (from the animal holding center at Shanghai JiaoTong University School of Medicine) in a healthy condition, aged 18 months with an average weight of 22.5 kg were used in this study, and all procedures were performed at the facility accredited by the Animal Care and Experiment Committee of Shanghai JiaoTong University School of Medicine. After anesthetization through intramuscular injection of ketamine (10 mg/kg), 5 mL of bone marrow aspirates were drawn from the iliac crests into a 10 mL syringe containing 6000 U of heparin. cBMSCs were isolated and cultured as previously described [10]. Briefly, mononuclear cells were separated by Percoll (1.063 g/mL, Sigma, St. Louise, MO) gradient centrifugation, and were plated in 100-mm dishes (Falcon, Franklin Lakes, NJ) at a density of 1.2  105 cells/cm2. Cells were cultured in the osteogenic medium (OM) (containing low-glucose Dulbecco’s modified Eagle’s medium (LG-DMEM), 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 mg/mL streptomycin sulfate, 10 8 mol/L dexamethasone, 10 mmol/L b-phosphoglycerol and 50 lmol/L L-2-ascorbic acid (all from Sigma)) at 37 °C with 5% CO2. The medium was changed after 48 h and later every 3 days. When cBMSCs reached 80–90% confluence, cells were detached with 0.25% trypsin–EDTA (Gibco),

Fig. 1. Structural properties of pDBM scaffolds. (A) Gross view of a pDBM scaffold with dimension of 5 mm in diameter and 1 mm in height. (B) A SEM image of the scaffold. The empty lacunae without cells after decelluarization were seen on pDBM (bar scale: 100 lm). (C) A 3D micro-CT image of the scaffold (bar scale: 1 mm).

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Zeiss, Germany) by staining viable cells with the green fluorescent dye Calcein AM and the red fluorescent dye Ethidium homodimer1 (LIVE/DEAD Viability/Cytotoxicity Assay Kit, Invitrogen Detection Technologies, USA) following the manufacturer’s protocol. Cell proliferation on the scaffolds within a period of 12 days was further determined by DNA assay as reported previously [6]. Briefly, TEBs collected at different time points were crushed for full lysis with proteinase K (Sigma) at 56 °C overnight. DNA content in the lysate was quantified spectrofluorometrically (523 nm) using Hoechst 33258 dye (Sigma) by correlating with a standard curve. To assess the in vitro osteogenesis of cBMSCs on the scaffolds, scanning electron microscopy (SEM, Philips XL-30, Amsterdam, Netherlands) examination of the TEB was carried out at 1 and 8 days after cell seeding as previously detailed [34], while ALP activity and OCN content were evaluated at 1, 4, 8, 16, 24 and 32 days after cell seeding as described previously [6]. The amounts of ALP and OCN produced by each sample were normalized to the total cell number of that sample determined by DNA assay, thereby allowing statistical comparisons to be made between different groups. The non-induced cBMSCs which were cultured in basic culture medium (BCM) (LG-DMEM supplemented with 10% FBS) and similarly seeded on the pDBM scaffolds, and the pDBM scaffolds without any cells but soaked under the same medium, were also measured to serve as controls. Vitreous cryopreservation and rewarming of TEB In the present study, a novel vitreous solution was developed and named as VS442 which consisted of 40% (v/v) dimethyl-sulfoxide (DMSO), 40% (v/v) EuroCollins (EC) solution and 20% (v/v) BCM. A commercialized vitreous medium, VS55 (a cocktail of 31.2% DMSO, 23% propylene glycol, 17.4% formamide and 28.4% EC solution in volume), was used as a control [7,23,30]. After being cultured for 8 days in OM, the TEB was transferred to freezing vials of 2 mL containing 0.5 mL VS442 or VS55 solution at 0 °C for 15 min, and then directly quenched into liquid nitrogen (LN2). Following vitrification for 7 days or 3 months, the cryovials were removed from the LN2 Dewar, and immediately immersed in a water bath of 37 °C with shaking until the vitreous solution turned completely from a glass-like state into liquid state. After

that, the TEB was removed from the cryoprotective agents, quickly transferred into fresh OM in a shaking incubator for 15 min at 37 °C, and further transferred into 24-well plates. One mL of OM was added into each well, and was then changed every 3 days. Cell viability and osteogenesis of TEB after vitreous cryopreservation Following the rewarming procedures, cell viability of TEB was visualized at days 1, 3 and 11 by fluorescent labeling as described above. Meantime, cell proliferation on the scaffolds was determined by DNA assay at days 1, 3, 5, 7, 9, 11, 13 and 15 as mentioned above. To detect in vitro osteogenesis of the current cryopreserved TEB, SEM observation was carried out at 1, 3 and 11 days after rewarming, while ALP activity and OCN content were assessed at 1, 3, 5, 7, 9, 11, 13 and 15 days after rewarming with the respective results normalized to the total cell numbers as detailed above. Statistical analysis Measurements for DNA assay, ALP activity and OCN content were carried out with n = 6 and expressed as means ± standard deviation (SD). All other experiments were conducted in triplicate. Data for these measurements were analyzed using paired Student’s t-test with the GraphPad Prism software. Statistically significant values were defined as p < 0.05. Results Growth and osteogenic differentiation of cBMSCs During in vitro expansion in OM, osteo-induced cBMSCs at P2 appeared as a relatively homogenous population which exhibited a fibroblast-like morphology, and proliferated rapidly with confluence being reached within 3 days (Fig. 2A). Osteogenic differentiation of cBMSCs was detected by positive intracellular expression of ALP (Fig. 2B) and deposition of calcified extracellular matrix (ECM) revealed by Alizarin Red staining (Fig. 2C). In addition, positive expression of OCN (Fig. 2D), OPN (Fig. 2E) and BSP (Fig. 2F) in

Fig. 2. In vitro osteogenesis of cBMSCs at P2. (A) Cells exhibited cuboidal osteoblast-like morphology after a 2-week osteogenic induction. (B) Positive ALP staining. (C) Alizarin Red staining indicating the ECM mineralization. The osteogenic differentiation was further verified by positive immunocytochemistry of OCN (D), OPN (E) and BSP (F) (Bar scales: 50 lm for A–C; 100 lm for D–F). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

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the osteogenic induced cells was revealed by immunocytochemistry staining. Proliferation and osteogenic differentiation of cBMSCs on scaffolds To observe the attachment, survival and growth of cBMSCs on the pDBM scaffold, TEB stained with fluorescent dyes of Calcein AM and Ethidium homodimer-1 was viewed under CLM. As shown in Fig. 3A, cells adhered well to the surface of scaffold in a dispersed way along the rim of pores after 1 day. After proliferation for 8 days, most of the pores were filled with clusters of viable cells characterized by green fluorescence. Moreover, no red-staining dead cells could be observed in the TEB at either 1 day or 8 days. By Hoechst 33258 assay, it was determined that the cell number in either osteo-induced group or non-induced group increased to a peak value at day 8 post-seeding and maintained at a plateau afterwards. Of note, cell number in osteo-induced group was significantly higher than that in non-induced group after 4 days (Fig. 3B). By SEM observation, it was found that cells attached and spread well on surface of scaffold with a few ECM deposited at day 1. When it came to day 8, surface of scaffold was almost totally covered with ECM (Fig. 4A). To further address whether the osteogenic phenotype of cBMSCs grown in the scaffold could be maintained, the expression of ALP and the release of OCN were measured at days 1, 4, 8, 16, 24 and 32 post-seeding, respectively. As shown in Fig. 4B and C, the expression of ALP and OCN secretion in the induced group kept on increasing after seeding, and no decline was found within 32 days. A significant elevated ALP activity and OCN secretion was detected in the induced group at each time point tested since day 8 as compared with the corresponding ones in the non-induced group in which expression of ALP and OCN remained almost undetectable (p < 0.05). Based on the above results, it was found that a simultaneous enhancement in cell proliferation and ECM deposition occurred since day 8. Thus, TEB being cultured in vitro for 8 days was chosen for subsequent vitreous cryopreservation. Cell viability of TEB after being cryopreserved in solutions of different vitreous formulae To investigate the survival and proliferative ability of cells after preservation, the cell number in TEB preserved in either VS442 or VS55 for 7 days was detected within the duration of 15 days postrewarm, respectively. By Hoechst 33258 assay, it was found that the cell number at day 1 post-rewarm reduced by 19% and 32%

1 day

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Osteogenesis of TEB after being cryopreserved in solutions of different vitreous formulae To further address whether osteogenic potential of cBMSCs in cryopreserved TEB could be maintained, mineralized ECM deposi-

B Cell number (×106/disc)

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in VS442 and VS55 groups, respectively, compared with that before preservation (2.64  106 cells per TEB at 8 days after cell seeding). A further decrease in the cell number was detected at day 3 postrewarm, reaching only 47% (VS442) and 46% (VS55) of those in samples prior to freezing, respectively. However, a constant increase in the cell number was observed in the VS442 group since that time point, reaching 90% of that of pre-cryopreservation at 11 days and maintained at this high level in the following assay days. In contrast, the cell number in the VS55 group kept on decreasing during the assay period after rewarming, reaching only 6% of that before cryopreservation. Moreover, a significant difference in the cell viability was observed between VS442 and VS55 groups at each time point since day 3 post-rewarming (Fig. 5A). The results of live/dead double staining were well correlated with the above Hoechst 33258 assay, that is a gradual increase in the amount of living cells occupied on the surface of pDBM scaffold in the VS442 group was observed after rewarming with the decrease in the number of dead cells. On the contrary, only redstained dead cells could be observed without obvious appearance of green-stained live cells in VS55 treated TEB at the same period (Fig. 5B). To further address whether cell viability was affected by the duration of cryopreservation via vitrification, preservation time of TEB was extended to 3 months and then the cell number was measured again after rewarming. As shown in Fig. 5A, survival and proliferation of cBMSCs cryopreserved for 3 months exhibited a similar increasing fashion in the cell number after remarming as that of cells preserved for 7 days in VS442 group. No significant difference in the cell viability was detected between the 3 months and 7 days preserved groups at each time point tested after rewarming. The cell number in the VS55 group which had been cryopreserved for 3 months kept on decreasing after rewarming as that in 7 days preserved group. There was no significant difference in the cell number between the 3 months and 7 days cryopreserved groups of VS55 treated. Similar to the phenomenon observed in the groups cryopreserved for 7 days, the cell number in VS442 group was maintained significantly higher since 3 days after rewarming than that in VS55 group after being cryopreserved for 3 months.

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Fig. 4. In vitro characterization of osteogenic differentiation of TEB. (A) SEM evaluation. Compared with day 1, dense ECM covered the pores of the scaffold at day 8 after cell seeding (bar scales: 400 lm). (B and C) Osteogenic differentiation of cBMSCs on pDBM scaffolds was evaluated by the expression of ALP and release of OCN at days 1, 4, 8, 16, 24 and 32 after seeding, respectively. pDBM scaffold alone cultured in OM served as a control (the error bars indicate SD for n = 4).

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Fig. 5. In vitro cellular activity for TEB following 7 days or 3 months of vitrification. (A) Cellular number measurements were obtained at days 1, 3, 5, 7, 9, 11, 13 and 15 postrewarm. More viable cells were found in VS442 groups compared to VS55 groups (p < 0.05). No significant difference in cell number was detected between 7-day groups and 3-month groups (p > 0.05; the error bars indicate SD for n = 4) both using VS442. (B) CLM images of TEB stained with Calcein AM (Green: Live) and Ethidium homodimer-1 (Red: Dead) after 1, 3 and 11 days of rewarming, respectively (bar scales: 250 mm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

tion was observed within 15 days after rewarming. According to the SEM observation, it was shown that surface of VS442 treated TEB (cryopreservation for 7 days) at days 1, 3 and 11 after rewarm-

ing was covered with a smooth layer of ECM with some minor fissures (Fig. 6A). No obvious difference in the fashion of ECM deposition was observed in VS442 treated TEB at 1, 3 and 11 days after

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Time of cultivation/d Fig. 6. In vitro osteogenic potential for TEB following 7 days or 3 months of vitrification. (A) SEM evaluation of cryopreserved TEB at 1, 3 and 11 days after rewarming, respectively (bar scales: 400 lm). (B and C) Osteogenic differentiation for TEB was evaluated by the expression of ALP and release of OCN at days 1, 3, 5, 7, 9, 11, 13 and 15 post-rewarm, respectively. Significantly increased osteogenesis was found in VS442 groups compared to VS55 groups. No significant difference in osteogenesis was detected between 7-day groups and 3-month groups (p > 0.05; the error bars indicate SD for n = 4).

rewarming. However, VS55 treated TEB displayed a more coarse appearance at 1, 3 and 11 days after rewarming, indicating the broken of ECM (Fig. 6A). More importantly, it was found that expression of ALP (Fig. 6B) and OCN secretion (Fig. 6C) in the VS442 cryopreserved group (7 days) kept on increasing after rewarming, reaching the original pre-cryopreservation level at day 9 and 7, respectively. However, those in the VS55 group decreased constantly and could no longer be detected at day 7 and day 11, respectively. Levels of ALP activity and OCN secretion in the VS442 group were significantly higher than those in the VS55 group at the same time point. To explore the effect of preservation time on the osteogenic potential of TEB, they were cryopreserved in either VS442 or VS55 for 3 months and ECM deposition was detected after rewarming. It was shown by SEM images that abundant ECM deposition was observed on the surface of VS442 preserved TEB at 1, 3 and 11 days after rewarming (Fig. 6A). However, only sparsely distributed ECM could be observed in VS55 preserved TEB (Fig. 6A). A similar increase in the amounts of ALP (Fig. 6B) and OCN (Fig. 6C) was detected after rewarming in VS442 group of 3 months to that of 7 days without significant difference at each time point tested. On the other hand, similar to those observed in the VS55 group of 7 days, the amounts of ALP and OCN in VS55 group of 3 months kept on decreasing and declined to a negligible level at 7 and 11 days after rewarming, respectively.

Discussion As one of the leading edge in tissue engineering, bone tissue engineering has shown promising potential in treating bone defects both in animal models [28,35] and in human bodies [4] in a variety of studies. However, cryopreservation of TEB, which plays

an important role in meeting the ‘‘ready-to-use” requirement in clinic, is just on the horizon. Though many cryopreservation procedures have been developed for osteoblasts and TEB in an attempt to meet such a requirement, the results are always unsatisfactory as ice formation was un-avoidable to cause cell damage and scaffold distortion by conventional cryopreservation approaches [7,31]. Recently, it has been well documented that vitreous cryopreservation is more superior to conventional freezing methods in preserving cells [16] and cell/scaffold complex [33] with elimination of the formation of ice crystals during cooling and warming processes. Thus, we explored the feasibility of vitreous cryopreservation of TEB composed of osteogenic cBMSCs and pDBM scaffold, and compared the effect of two cryoprotectants (VS55 and VS442) on the viability and osteogenic potential of TEB. Choosing a suitable cryoprotective agent which was formulated to replace intracellular water by forming an amorphous state to eliminate ice formation, is the most critical issue concerning the success of vitreous cryopreservation of cells and tissues [15]. Cryoprotective chemicals can be basically classified as penetrating agents and non-penetrating ones [11]. As a conventional vitreous solution, VS55 was successfully used to cryopreserve cells [18], tissues [31] and organs [23]. Furthermore, VS55 was also shown to be effective in vitreous cryopreserving tissue engineered blood vessel [7] and pancreatic substitute [30]. However, in this study the cellular viability and mineralization capacity of cryopreserved TEB using VS55 were observed to keep on decreasing to a trivial level within the duration of 15 days after rewarming. This result could be partially explained by the high toxicity to cells resulting from three penetrating agents of DMSO, 1,2-propanediol and formamide accounting for 71.6% of the total volume in VS55. In a study by Liu et al. [18], it was found that lowering the concentration of VS55 from full strength to 90% resulted in a significant increase in the cellular viability when vitreous cryopreserving osteoblasts. Thus,

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to minimize the toxicity resulting from cryoprotectants, it is reasonable to develop a novel formula with lower concentrations of penetrating agents while maintaining a desired cryoprotective effect. We thus designed a novel vitreous solution: VS442 in which the volumes of DMSO, EC and BCM were adjusted to a volumetric ratio of 4:4:2. Since the addition of highly water soluble DMSO results in an osmolality both outside and inside cells to prevent the formation of intracellular ice crystals, DMSO is widely recommended as a cryoprotectant for cell/scaffold constructs [2]. Moreover, it was shown that the glass forming effect of DMSO is superior to that of formamide [8] and the toxicity of DMSO is less than that of 1,2propanediol [18]. Hence, DMSO was chosen as the cryoprotectant in this study. However, it was found that its ability of penetrating tissues and cell/scaffold complex in a large mass was quite limited at concentrations ranging from 8% to 20% [7,11]. Thereafter, a concentration of 40% in the vitreous formula was adopted. According to Liu et al. [18], among different concentrations lower or higher than 40%, DMSO at 40% achieved a highest cellular viability when it was used to vitreous cryopreserve osteoblasts. Furthermore, to mimic the composition and concentrations of intracellular electrolytes [9], EC solution was chosen as another component in a ratio of 1:1 (v/v) to DMSO, similar to that (1:1.1) in VS55. Although FBS-based medium is widely used in conventional slow freezing methods [29], its effect on vitreous preservation is controversial. In consideration of the possible contamination from an animal protein in clinic [14], we originally designed the rest part of VS442 by supplementing 20% deionized water. However, the viability of cells after rewarming was far from satisfactory (data not shown). When 20% BCM was added to replace deionized water, cellular viability was remarkably enhanced after rewarming. We speculated that cells on the scaffold might still undergo some metabolic activities in the process of preconditioning, so serum and nutrients in the medium were necessary for their survival. Compared with that of TEB cryopreserved in VS55, the cellular viability of TEB banked in VS442 was significantly higher at each time point tested within the duration of 15 days after rewarming. More importantly, it was shown that, after a decrease in the initial 3 days, the cellular viability in VS442 treated TEB kept on increasing and recovered to 90% of that in pre-cryopreserved TEB at 11 days. However, viability of cBMSCs stored in VS55 exhibited a declining trend down to an undetectable level at 15 days after rewarming. These results indicated that VS442 is more suitable than VS55 solution to cryopreserve current TEB. However, the 47% recovery in cellular viability in the VS442 group at 3 days after rewarming is still far from the ‘‘ready-to-use” requirement for clinical applications. Two reasons may account for this low cellular viability. First, the concentration of 40% DMSO in the vitreous solution is relatively high as such a level of DMSO is considerably toxic to cells [14] resulting in cell death during the vitrification process. Second, due to the relatively larger volume of TEB compared with that of cell suspensions, the critical cooling rate is not always achievable at the center of TEB leading to a coexistence of vitrified and crystallized state [23,25]. This would cause damage to cells reside in scaffold. The complicated thermophysical properties of scaffold may also influence the efficacy of cryopreservation either by freezing or by vitrification [27]. It was reported that when rabbit osteoblast-like cells were seeded on thin poly (lactide-co-glycolide) films, cellular viability decreased approximately 50% by conventional cryopreservation [12]. It was found by Liu and McGrath [17] that, when osteoblast/hydroxyapatite complex was cryopreserved in VEG with ice blockers, viability of cells was reduced to less than 30% of that before vitreous preservation. Osteogenic differentiation of BMSCs after being cryopreserved by vitrification is one of the key issues judging the success of bone

regeneration via a tissue engineering approach. As a marker indicating early osteogenic differentiation, ALP was shown to regulate organic or inorganic phosphate metabolism via the hydrolyzation of phosphate esters, and functioned as a plasma membrane transporter for inorganic phosphates. Osteocalcin is also an established indicator of the osteogenic differentiation and synthesized only by mature osteoblasts to bind both collagen and calcium in the ECM of bone tissue. Thus, expression of ALP and deposition of OCN after rewarming was detected quantitatively in both VS442 and VS55 treated groups. It was found that the level of ALP expression and OCN deposition in the VS442 group was always higher than those in the VS55 group since 3 days after rewarming. Moreover, similar to the declining trend of cellular viability, ALP expression and OCN deposition in the VS55 group also decreased rapidly to a negligible level at 7 and 11 days after rewarming, respectively. More importantly, the level of ALP expression and OCN deposition in the VS442 group recovered to a similar degree as those respective ones of pre-cooling at day 9 and day 7 in culture, respectively. Thus, the novel VS442 solution is demonstrated to be superior to VS55 in not only maintaining cellular viability but also osteogenic capacity of TEB after vitreous cryopreservation. Furthermore, by comparing the proliferation and osteogenesis of TEB cryopreserved in VS442 for 7 days and 3 months, it was shown that the duration of preservation in VS442 did not have a significant impact on the survival and osteogenic potential of TEB. It is thus evident that when cells are in the LN2 storage, molecular motion is significantly reduced and thermally driven reactions might not occur within a relative period. In the current study, we have developed a novel vitreous solution named as VS442 which contained 40% DMSO, 40% EC and 20% BCM for vitreous cryopreserving constructs of osteogenic differentiated cBMSCs and pDBM scaffold. The VS442 solution was demonstrated to be more efficient for vitreous cryopreservation of TEB in maintaining both cellular viability and osteogenic function compared with the commonly used one, VS55. It was also found that elongating the duration of vitreous preservation in VS442 from 7 days to 3 months had no significant influence on the survival and osteogenic potential of cBMSCs seeded in pDBM scaffold. References [1] E. Arnaud, C. De Pollak, A. Meunier, L. Sedel, C. Damien, H. Petite, Osteogenesis with coral is increased by BMP and BMC in a rat cranioplasty, Biomaterials 20 (1999) 1909–1918. [2] A.M. Bakken, Cryopreserving human peripheral blood progenitor cells, Curr. Stem. Cell. Res. Ther. 1 (2006) 47–54. [3] G. Bhakta, K.H. Lee, R. Magalhães, F. Wen, S.S. Gouk, D.W. Hutmacher, L.L. Kuleshova, Cryopreservation of alginate-fibrin beads involving bone marrow derived mesenchymal stromal cells by vitrification, Biomaterials 30 (2009) 336–343. [4] F. Casabona, I. Martin, A. Muraglia, P. Berrino, P. Santi, R. Cancedda, R. Quarto, Prefabricated engineered bone flaps: an experimental model of tissue reconstruction in plastic surgery, Plast. Reconstr. Surg. 101 (1998) 577–581. [5] L. Cui, D. Li, X. Liu, F. Chen, W. Liu, Y. Cao, Experimental study of partially demineralized bone matrix as bone tissue engineering scaffold, Key Eng. Mater. 288–289 (2005) 63–66. [6] L. Cui, B. Liu, G. Liu, W. Zhang, L. Cen, J. Sun, S. Yin, W. Liu, Y. Cao, Repair of cranial bone defects with adipose derived stem cells and coral scaffold in a canine model, Biomaterials 28 (2007) 5477–5486. [7] S.L. Dahl, Z. Chen, A.K. Solan, K.G. Brockbank, L.E. Niklason, Y.C. Song, Feasibility of vitrification as a storage method for tissue-engineered blood vessels, Tissue Eng. 12 (2006) 291–300. [8] G.M. Fahy, B. Wowk, J. Wu, S. Paynter, Improved vitrification solutions based on the predictability of vitrification solution toxicity, Cryobiology 48 (2004) 22–35. [9] W. Jassem, T. Armeni, J.L. Quiles, S. Bompadre, G. Principato, M. Battino, Protection of mitochondria during cold storage of liver and following transplantation: comparison of the two solutions, University of Wisconsin and Eurocollins, J. Bioenerg. Biomembr. 38 (2006) 49–55. [10] S. Kadiyala, R.G. Young, M.A. Thiede, S.P. Bruder, Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro, Cell Transplant. 6 (1997) 125–134.

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