Highlights
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Developed a novel injectable cell delivery system based on iPSMSC encapsulation in microbeads in a CPC paste.
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The first report on cranial bone reconstruction using self-setting microencapsulated iPSMSC-CPC paste in vivo in an animal model.
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2–3 fold increase in bone regeneration in vivo via OS-iPSMSCs and BMP2-iPSMSCs compared to CPC control without cell delivery.
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Cell-encapsulating-CPC constructs accelerated the degradation of the scaffold in vivo by 3–6 fold compared to CPC control.
Abstract
Objectives
Calcium phosphate cements (CPCs) are promising for dental and craniofacial repairs. The objectives of this study were to: (1) develop an injectable cell delivery system based on encapsulation of induced pluripotent stem cell-derived mesenchymal stem cells (iPSMSCs) in microbeads; (2) develop a novel tissue engineered construct by dispersing iPSMSC-microbeads in CPC to investigate bone regeneration in an animal model for the first time.
Methods
iPSMSCs were pre-osteoinduced for 2 weeks (OS-iPSMSCs), or transduced with bone morphogenetic protein-2 (BMP2-iPSMSCs). Cells were encapsulated in fast-degradable alginate microbeads. Microbeads were mixed with CPC paste and filled into cranial defects in nude rats. Four groups were tested: (1) CPC-microbeads without cells (CPC control); (2) CPC-microbeads-iPSMSCs (CPC-iPSMSCs); (3) CPC-microbeads-OS-iPSMSCs (CPC-OS-iPSMSCs); (4) CPC-microbeads-BMP2-iPSMSCs (CPC-BMP2-iPSMSCs).
Results
Cells maintained good viability inside microbeads after injection. The microbeads were able to release the cells which had more than 10-fold increase in live cell density from 1 to 14 days. The cells exhibited up-regulation of osteogenic markers and deposition of minerals. In vivo , new bone area fraction (mean ± SD; n = 5) for CPC-iPSMSCs group was (22.5 ± 7.6)%. New bone area fractions were (38.9 ± 18.4)% and (44.7 ± 22.8)% for CPC-OS-iPSMSCs group and CPC-BMP2-iPSMSCs group, respectively, 2–3 times the (15.6 ± 11.2)% in CPC control at 12 weeks ( p < 0.05). Cell-CPC constructs accelerated scaffold resorption, with CPC-BMP2-iPSMSCs having remaining scaffold material that was 7-fold less than CPC control.
Significance
Novel injectable CPC-microbead-cell constructs promoted bone regeneration, with OS-iPSMSCs and BMP2-iPSMSCs having 2–3 fold the new bone of CPC control. Cell delivery accelerated scaffold resorption, with CPC-BMP2-iPSMSC having remaining scaffold material that was 7-fold less than CPC control. Therefore, CPC-microbead-iPSMSC is a promising injectable material for orthopedic, dental and craniofacial bone regenerations.
1
Introduction
The generation of induced pluripotent stem cells (iPSCs) is an exciting discovery in the field of cell-based therapy . Through expression of a small combination of transcription factors, somatic cells can be converted into an embryonic state, thus, exhibiting tremendous possibilities in treatment of various diseases. IPSCs represent an enormous source of patient-specific stem cells derived from plentiful and easily accessible tissues like skin, hair and fat, etc . Development of virus-free and vector-free reprogramming technologies reduces the chance of virally-induced tumor formation, thus provides optimism for clinical applications of iPSCs . When transplanted back into the patients, iPSCs need to be induced into high-quality progenitor cells like mesenchymal stem cells (MSCs), or fully-differentiated homogenous mature cells to circumvent the risk of teratoma formation caused by undifferentiated cells contaminating the final products . It has been reported that iPSC-derived MSCs (iPSMSCs) exhibited a higher proliferative capability than bone marrow MSCs (BMSCs) , and are less tumorigenic than undifferentiated iPSCs and BMSCs . Thus in the field of bone tissue engineering, iPSMSCs escalate the hope especially for patients with compromised health conditions whose autologous BMSCs are no longer vibrant for tissue repair and regeneration .
Calcium phosphate cements are promising bone substitutes with excellent bioactivity, biocompatibility and osteoconductivity . These materials stand out as injectable bone cements owing to their self-setting and in situ -hardening capabilities . One such cement consisted of tetracalcium phosphate [TTCP: Ca 4 (PO 4 ) 2 O] and dicalcium phosphate (DCPA: CaHPO 4 ), and was referred to as CPC . The CPC powder can be mixed with an aqueous liquid to form a paste that can be injected or sculpted during surgery to conform to the defects in hard tissues. CPC was approved in 1996 by the Food and Drug Administration for repairing craniofacial defects in humans, thus becoming the first CPC available for clinical use .
Our previous studies enhanced the mechanical, physical and biological properties of CPC through the introduction of absorbable fibers , chitosan , mannitol porogen , gas-foaming agents , alginate microbeads , and biofunctionalization . These approaches improved the CPC’s mechanical strength, setting time, degradability, macroporosity, cell attachment, and delivery of cells and growth factors. Potential dental and craniofacial applications of an improved CPC include periodontal bone lesion repair, socket preservation, maxillary sinus floor elevation, augmentation of deficient implant sites, ridge augmentation, as well as other dental and orthopedic applications .
Our recent study showed that osteoinduced iPSMSCs seeded on pre-formed CPC scaffolds in rat cranial defects had comparable in vivo bone regeneration capability to BMSCs and umbilical cord MSCs (UCMSCs), but significantly higher than CPC control . No teratoma was found during a 12 weeks observation . Although it was promising in supporting iPSMSCs’ role in prompting bone regeneration efficiency of CPCs, cells were only loaded on one side of the scaffolds. This type of static cell seeding method has limitations of low seeding efficiency and minimal cell penetration into scaffold, leading to non-uniform distribution of cells and subsequently compromised regeneration in vivo . To address these problems, in the present study, alginate microbeads were used as cell delivery vehicles to protect the encapsulated cells during CPC paste mixing, injection and setting reactions. The CPC-microbead-constructs can be readily injected or placed into bone defects with minimal invasion and intimate adaptation to complex defect shapes . Alginate has been selected because it is non-cytotoxic and can form an ionically cross-linked network under mild conditions producing no detrimental effects to cells . To promote alginate degradation and subsequent cell release, fast-degradable alginate-fibrin microbeads were fabricated following a previous study . Furthermore, to enhance osteogenicity, iPSMSCs were either pre-osteoinduced for 2 weeks (OS-iPSMSCs), or transduced with bone morphogenic protein-2 (BMP2) gene (BMP2-iPSMSCs).
The aims of this study were to: (1) develop a novel injectable cell delivery system based on iPSMSC encapsulation in alginate microbeads and investigate cell viability, proliferation and osteogenic differentiation; and (2) develop a novel tissue engineered construct by dispersing iPSMSC-microbeads in CPC and investigate bone regeneration in vivo . The following hypotheses were tested: (1) microencapsulation and injection would not harm the encapsulated iPSMSCs; (2) iPSMSCs released from fast-degradable microbeads could proliferate and differentiate into osteogenic lineage; (3) cell-encapsulating-microbeads can induce osteogenic differentiation of co-cultured BMSCs; and (4) pre-osteogenic differentiation and BMP2 transduction would promote bone regeneration of iPSMSCs in CPC constructs in vivo .
2
Materials and methods
2.1
Derivation of MSCs from iPSCs
Human iPSC BC1 line was maintained on mitotically-inactivated murine embryonic fibroblasts (MEF) feeder . IPSCs were detached from MEF and dissociated into clumps by treatment with collagenase type IV. The dissociated iPSC clumps were collected, resuspended, and transferred to ultra-low attachment cell culture flasks (Corning, Corning, NY). After 10 days (d), EBs were transferred onto 0.1% gelatin coated culture dishes. Cells growing from EBs were cultured and upon 70% confluence, outgrowth cells were selectively isolated using cell scrapers and sub-cultured in MSC growth media, which consisted of low glucose Dulbecco’s modified Eagle’s media (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco). The differentiated cells were passaged until a homogeneous fibroblastic morphology appeared and were termed iPSMSCs. Our previous study confirmed that iPSMSCs generated from this method expressed surface markers of MSCs (CD29, CD44, CD166, CD73), and were negative for typical hematopoietic (CD34), endothelial (CD31) and pluripotent markers (TRA-1-81 and OCT 3/4). The iPSMSCs could differentiate into three characteristic mesenchymal lineages including osteoblasts, adipocytes and chondrocytes . The iPSMSCs used in this study have also been tested positive for CD29, CD44, CD166, CD73, but negative for CD34, CD31, TRA-1-81 and OCT 3/4. The 3–5th passage iPSMSCs were used in the following experiments.
2.2
BMP2 gene transduction of iPSMSCs
Lentiviruses with human BMP2 (GenTarget, San Diego, CA) were used for gene transduction. Passage 3 iPSMSCs were exposed to 15 multiplicity of infection of BMP2 lentiviruses for 3 d. These cells are referred to as BMP2-iPSMSCs. Our previous study showed that transduction efficacy was more than 68.8%, and BMP2 gene and protein were stably expressed from passage 5 to 8. Cell proliferation was comparable to iPSMSCs while osteogenic differentiation was enhanced in BMP2-iPSMSCs .
2.3
Synthesis of alginate microbeads with cell encapsulation
Alginate (64% guluronic acid, MW = 75,000–220,000 g/mol, ProNova, Oslo, Norway) was oxidized at 7.5% oxidation to increase its degradability . The oxidized alginate was dissolved in saline at a concentration of 1.2%. Fibrinogen from bovine plasma (Sigma) was added at a concentration of 0.1% to the alginate solution to further accelerate alginate degradation . Cells were added to the alginate-fibrinogen solution at a density of 1 × 10 6 cells/mL. The cell solution was loaded into a syringe which was connected to a bead generating device (Var J1, Nisco, Zurich, Switzerland). Nitrogen gas at a pressure of 8 psi was established to form a coaxial air flow to break up alginate droplets. The droplets fell into a well containing 125 mL of 100 mmol/L calcium chloride plus 125 NIH units of thrombin (Sigma). Calcium chloride caused alginate to crosslink, while the reaction between fibrinogen and thrombin produced fibrin. This yielded cell-encapsulating-alginate-fibrin-microbeads with diameters of about 100–500 μm, with a mean of 311 μm . Three groups of cell-encapsulating-microbeads were produced. (1) iPSMSCs. (2) Pre-osteoinduced iPSMSCs (OS-iPSMSCs). These were cells that underwent 2 w osteogenic differentiation in osteogenic media (OS media) which consisted of MSC growth media supplemented with 100 nM dexamethasone, 10 mM β-glycerophosphate, 0.05 mM ascorbic acid, and 10 nM 1а,25-dihydroxyvitaminD3 (Sigma). (3) BMP2-iPSMSCs.
2.4
Cell viability and proliferation
Cell-encapsulating-microbeads (50 μL) were added to each well of 24 well-plates with 1 mL MSC growth media. Twelve wells were prepared at each time-point (1, 3, 7 and 14 d), with six wells for live/dead staining, and six wells for cell proliferation assay. Cells were stained with a live/dead kit (Invitrogen) and observed via epifluorescence microscopy (Eclipse TE-2000S, Nikon, Melville, NY). Two images were taken at random locations for each sample, with six samples yielding 12 images for iPSMSC-microbeads at each time point. Live and dead cells were counted separately in green or red channels via Image J2 software. After subtracting the background with an automatic threshold, the images were converted to binary images. Objects in a binary image that overlapped each other were corrected by using the watershed separation function of the software. Once the image has been segmented, the menu command “Analyze/Analyze particles” was used to obtain various information regarding particle size and numbers. The percentage of live cells P = number of live cells/(number of live cells + number of dead cells) × 100%. Live cell density D = number of live cells in the image/image area .
Cell counting kit (CCK-8; Dojindo, Tokyo, Japan) was used to evaluate cell proliferation from 1 to 14 d. To investigate if the injection process would harm the encapsulated cells, iPSMSC viability at 1 d was compared between: (1) cells in microbeads without injection, and (2) cells in microbeads after injection from a 10 mL syringe (Free-Flo, Kerr, Romulus, MI) with a tip opening of 2.7 mm which was similar to the inner diameter of a 10-gauge needle .
2.5
Osteogenic differentiation and mineralization of cell-encapsulating-microbeads
Three types of microbeads were cultured in OS media for 2 weeks. At 1, 7 and 14 d, TaqMan gene expression kits were used to quantify targeted genes on human alkaline phosphatase (ALP, Hs00758162_ml, RefSeq NM_000478.4, assay location: 1120, amplicon length: 84; RefSeq NM_001127501.2, assay location: 955, amplicon length: 84; RefSeq NM_001177520.1, assay location: 839, amplicon length: 84), Runt-related transcription factor (RUNX2, Hs00231692_ml, RefSeq NM_001015051.3, assay location: 900, amplicon length: 116; RefSeq NM_001024630.3, assay location: 900, amplicon length: 116; RefSeq NM_001278478.1, assay location: 648, amplicon length: 116), collagen type-I, alpha 1 (COL1A1, Hs00164004_m1, RefSeq NM_000088.3, assay location: 230, amplicon length: 66), osteocalcin (OCN, Hs01587814_g1, RefSeq NM_199173.4, assay location 177, amplicon length: 116), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs99999905, RefSeq NM_002046.4, assay location: 229, amplicon length: 122). Relative expression was evaluated using the 2 −ΔΔCt method and normalized by the Ct of the housekeeping gene GAPDH. Ct of iPSMSCs in growth media cultured on tissue culture polystyrene (TCPS) for 1 d served as the calibrator .
At 1, 7 and 14 d, cell-encapsulating-microbeads were stained with Alizarin Red S (ARS, Millipore, Billerica, MA). The ARS concentration was measured by the Osteogenesis Quantitation Kit (Millipore) .
2.6
Osteogenic differentiation of BMSCs in co-culture with cell-encapsulating-microbeads
Co-culture of cell-encapsulating-microbeads and non-encapsulated BMSCs was conducted in a transwell system (BD Biosciences), which allowed the sharing of conditioned media without cell–cell contact. Non-encapsulated BMSCs were seeded into the lower compartment of 12-well plates at 5 × 10 4 cells/well, and 50 μL cell-encapsulating-microbeads were added to the upper compartments for co-culture in MSC growth media for 2 weeks. At 1, 7 and 14 d, TaqMan gene expression kits were used to quantify targeted genes on human ALP (Hs00758162_ml), RUNX2 (Hs00231692_ml), OCN (Hs01587814_g1), osteopontin (OPN, Hs00959010_m1, RefSeq NM_000582.2, assay location: 657, amplicon length: 84; RefSeq NM_001040058.1, assay location: 699, amplicon length: 84; RefSeq NM_001040060.1, assay location: 618, amplicon length: 84; RefSeq NM_001251829.1, assay location: 576, amplicon length: 84; RefSeq NM_001251830.1, assay location: 888, amplicon length: 84) and GAPDH (Hs99999905). Gene expression was evaluated using the 2 −ΔΔCt method as described above.
2.7
Fabrication of CPC scaffolds
CPC powder was prepared following a previous study . Briefly, TTCP [Ca 4 (PO 4 ) 2 O] was synthesized using DCPA (CaHPO 4 ) and calcium carbonate (both from J.T. Baker, Philipsburg, NJ) which were mixed and heated at 1500 °C for 6 h in a furnace (Model 51333, Lindberg, Watertown, WI). The heated mixture was quenched to room temperature in a desicator, ground in a ball mill (Retsch PM4, Brinkman, NY) and sieved to obtain TTCP powder with a median particle size of 5 μm. The commercial DCPA powder was ground for 24 h in the ball mill in 95% ethanol and sieved to obtain a median particle size of approximately 1 μm. Then the TTCP and DCPA powders at 1:3 molar ratio were thoroughly mixed in a micromill (Bel-Alert Products, Pequannock, NJ) to form the CPC powder . The microbeads were incorporated into CPC at 50% by volume. Degradable suture fibers (Ethicon, Somerville, NJ) were cut into 3 mm filaments and added into CPC at 10% by volume for mechanical reinforcement. The CPC liquid was 0.2 M Na 2 HPO 4 . A powder: liquid ratio of 2:1 was used to form a flowable CPC paste.
2.8
In vivo double cranial bone defects in rats
Double cranial defects of 5 mm each were created in 8-w-old male athymic nude rats (Hsd:RH-Fox1 mu , 200–250 g, Harlan, Indianapolis, IN) following a protocol approved by the University of Maryland Baltimore (IACUC # 0909014) and NIH animal-care guidelines. Briefly, under general anesthesia, two full-thickness 5 mm defects were made in the calvarium under continuous saline irrigation . CPC paste was mixed at the time of implantation and set in situ to fill the bone defects. Four groups were tested: (1) CPC-microbeads without cells (CPC control); (2) CPC-microbeads-iPSMSCs (CPC-iPSMSCs); (3) CPC-microbeads-OS-iPSMSCs (CPC-OS-iPSMSCs); (4) CPC-microbeads-BMP2-iPSMSCs (CPC-BMP2-iPSMSCs). Grafts were harvested after 12 weeks ( n = 5).
2.9
Histomorphometric analyses
Specimens were decalcified and embedded in paraffin. The central part of the implant and defect was cut into 5 μm-thick sections and stained with hematoxylin and eosin (HE) and Masson’s Trichrome (MT). New bone area, residual material area and total defect area was measured within the boundaries of defects in each section by Image Pro Plus Software (Media Cybernetics, Carlsbad, CA). New bone area fraction (NBAF) was calculated as the new bone area divided by total defect area ( n = 5). CPC scaffold residual material area fraction (RMAF) was calculated as the residual scaffold material area divided by total defect area ( n = 5).
2.10
Identification of encapsulated cells by immunohistochemistry (IHC)
Human origin of engineered bone constructs following in vivo implantation was detected using mouse monoclonal anti-human nuclei antibodies (Millipore). Tissue sections were deparaffinized with xylene, and rehydrated with a graded series of ethanol washes. The epitopes were recovered by incubation in citrate buffer at 70 °C for 40 min, and the endogenous peroxidase activity was blocked with 3% H 2 O 2 . The slides were then blocked with 1% BSA for 30 min to suppress nonspecific staining and stained with primary antibodies (1:50) overnight in a humidified environment. The specimens were subsequently incubated with secondary antibody against mouse IgG (1:500) for 30 min at 37 °C. Incubation was followed by streptavidin-HRP and diaminobenzidine (DAB) substrate, and counterstaining with hematoxylin solution. Negative controls were performed following the same procedures but without primary antibody incubation.
2.11
Statistical analyses
Statistical analyses were performed using Statistical Package for the Social Sciences (SPSS 19.0, Chicago, IL). All data were expressed as the mean value ± standard deviation (SD). All in vitro tests were independently repeated three times, with at least triplicate cultures for each condition. Statistical significance was analyzed by using the one-way analyses of variance (ANOVA) and Student–Newman–Keuls test. A confidence level of 95% ( p < 0.05) was considered significant.
2
Materials and methods
2.1
Derivation of MSCs from iPSCs
Human iPSC BC1 line was maintained on mitotically-inactivated murine embryonic fibroblasts (MEF) feeder . IPSCs were detached from MEF and dissociated into clumps by treatment with collagenase type IV. The dissociated iPSC clumps were collected, resuspended, and transferred to ultra-low attachment cell culture flasks (Corning, Corning, NY). After 10 days (d), EBs were transferred onto 0.1% gelatin coated culture dishes. Cells growing from EBs were cultured and upon 70% confluence, outgrowth cells were selectively isolated using cell scrapers and sub-cultured in MSC growth media, which consisted of low glucose Dulbecco’s modified Eagle’s media (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco). The differentiated cells were passaged until a homogeneous fibroblastic morphology appeared and were termed iPSMSCs. Our previous study confirmed that iPSMSCs generated from this method expressed surface markers of MSCs (CD29, CD44, CD166, CD73), and were negative for typical hematopoietic (CD34), endothelial (CD31) and pluripotent markers (TRA-1-81 and OCT 3/4). The iPSMSCs could differentiate into three characteristic mesenchymal lineages including osteoblasts, adipocytes and chondrocytes . The iPSMSCs used in this study have also been tested positive for CD29, CD44, CD166, CD73, but negative for CD34, CD31, TRA-1-81 and OCT 3/4. The 3–5th passage iPSMSCs were used in the following experiments.
2.2
BMP2 gene transduction of iPSMSCs
Lentiviruses with human BMP2 (GenTarget, San Diego, CA) were used for gene transduction. Passage 3 iPSMSCs were exposed to 15 multiplicity of infection of BMP2 lentiviruses for 3 d. These cells are referred to as BMP2-iPSMSCs. Our previous study showed that transduction efficacy was more than 68.8%, and BMP2 gene and protein were stably expressed from passage 5 to 8. Cell proliferation was comparable to iPSMSCs while osteogenic differentiation was enhanced in BMP2-iPSMSCs .
2.3
Synthesis of alginate microbeads with cell encapsulation
Alginate (64% guluronic acid, MW = 75,000–220,000 g/mol, ProNova, Oslo, Norway) was oxidized at 7.5% oxidation to increase its degradability . The oxidized alginate was dissolved in saline at a concentration of 1.2%. Fibrinogen from bovine plasma (Sigma) was added at a concentration of 0.1% to the alginate solution to further accelerate alginate degradation . Cells were added to the alginate-fibrinogen solution at a density of 1 × 10 6 cells/mL. The cell solution was loaded into a syringe which was connected to a bead generating device (Var J1, Nisco, Zurich, Switzerland). Nitrogen gas at a pressure of 8 psi was established to form a coaxial air flow to break up alginate droplets. The droplets fell into a well containing 125 mL of 100 mmol/L calcium chloride plus 125 NIH units of thrombin (Sigma). Calcium chloride caused alginate to crosslink, while the reaction between fibrinogen and thrombin produced fibrin. This yielded cell-encapsulating-alginate-fibrin-microbeads with diameters of about 100–500 μm, with a mean of 311 μm . Three groups of cell-encapsulating-microbeads were produced. (1) iPSMSCs. (2) Pre-osteoinduced iPSMSCs (OS-iPSMSCs). These were cells that underwent 2 w osteogenic differentiation in osteogenic media (OS media) which consisted of MSC growth media supplemented with 100 nM dexamethasone, 10 mM β-glycerophosphate, 0.05 mM ascorbic acid, and 10 nM 1а,25-dihydroxyvitaminD3 (Sigma). (3) BMP2-iPSMSCs.
2.4
Cell viability and proliferation
Cell-encapsulating-microbeads (50 μL) were added to each well of 24 well-plates with 1 mL MSC growth media. Twelve wells were prepared at each time-point (1, 3, 7 and 14 d), with six wells for live/dead staining, and six wells for cell proliferation assay. Cells were stained with a live/dead kit (Invitrogen) and observed via epifluorescence microscopy (Eclipse TE-2000S, Nikon, Melville, NY). Two images were taken at random locations for each sample, with six samples yielding 12 images for iPSMSC-microbeads at each time point. Live and dead cells were counted separately in green or red channels via Image J2 software. After subtracting the background with an automatic threshold, the images were converted to binary images. Objects in a binary image that overlapped each other were corrected by using the watershed separation function of the software. Once the image has been segmented, the menu command “Analyze/Analyze particles” was used to obtain various information regarding particle size and numbers. The percentage of live cells P = number of live cells/(number of live cells + number of dead cells) × 100%. Live cell density D = number of live cells in the image/image area .
Cell counting kit (CCK-8; Dojindo, Tokyo, Japan) was used to evaluate cell proliferation from 1 to 14 d. To investigate if the injection process would harm the encapsulated cells, iPSMSC viability at 1 d was compared between: (1) cells in microbeads without injection, and (2) cells in microbeads after injection from a 10 mL syringe (Free-Flo, Kerr, Romulus, MI) with a tip opening of 2.7 mm which was similar to the inner diameter of a 10-gauge needle .
2.5
Osteogenic differentiation and mineralization of cell-encapsulating-microbeads
Three types of microbeads were cultured in OS media for 2 weeks. At 1, 7 and 14 d, TaqMan gene expression kits were used to quantify targeted genes on human alkaline phosphatase (ALP, Hs00758162_ml, RefSeq NM_000478.4, assay location: 1120, amplicon length: 84; RefSeq NM_001127501.2, assay location: 955, amplicon length: 84; RefSeq NM_001177520.1, assay location: 839, amplicon length: 84), Runt-related transcription factor (RUNX2, Hs00231692_ml, RefSeq NM_001015051.3, assay location: 900, amplicon length: 116; RefSeq NM_001024630.3, assay location: 900, amplicon length: 116; RefSeq NM_001278478.1, assay location: 648, amplicon length: 116), collagen type-I, alpha 1 (COL1A1, Hs00164004_m1, RefSeq NM_000088.3, assay location: 230, amplicon length: 66), osteocalcin (OCN, Hs01587814_g1, RefSeq NM_199173.4, assay location 177, amplicon length: 116), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs99999905, RefSeq NM_002046.4, assay location: 229, amplicon length: 122). Relative expression was evaluated using the 2 −ΔΔCt method and normalized by the Ct of the housekeeping gene GAPDH. Ct of iPSMSCs in growth media cultured on tissue culture polystyrene (TCPS) for 1 d served as the calibrator .
At 1, 7 and 14 d, cell-encapsulating-microbeads were stained with Alizarin Red S (ARS, Millipore, Billerica, MA). The ARS concentration was measured by the Osteogenesis Quantitation Kit (Millipore) .
2.6
Osteogenic differentiation of BMSCs in co-culture with cell-encapsulating-microbeads
Co-culture of cell-encapsulating-microbeads and non-encapsulated BMSCs was conducted in a transwell system (BD Biosciences), which allowed the sharing of conditioned media without cell–cell contact. Non-encapsulated BMSCs were seeded into the lower compartment of 12-well plates at 5 × 10 4 cells/well, and 50 μL cell-encapsulating-microbeads were added to the upper compartments for co-culture in MSC growth media for 2 weeks. At 1, 7 and 14 d, TaqMan gene expression kits were used to quantify targeted genes on human ALP (Hs00758162_ml), RUNX2 (Hs00231692_ml), OCN (Hs01587814_g1), osteopontin (OPN, Hs00959010_m1, RefSeq NM_000582.2, assay location: 657, amplicon length: 84; RefSeq NM_001040058.1, assay location: 699, amplicon length: 84; RefSeq NM_001040060.1, assay location: 618, amplicon length: 84; RefSeq NM_001251829.1, assay location: 576, amplicon length: 84; RefSeq NM_001251830.1, assay location: 888, amplicon length: 84) and GAPDH (Hs99999905). Gene expression was evaluated using the 2 −ΔΔCt method as described above.
2.7
Fabrication of CPC scaffolds
CPC powder was prepared following a previous study . Briefly, TTCP [Ca 4 (PO 4 ) 2 O] was synthesized using DCPA (CaHPO 4 ) and calcium carbonate (both from J.T. Baker, Philipsburg, NJ) which were mixed and heated at 1500 °C for 6 h in a furnace (Model 51333, Lindberg, Watertown, WI). The heated mixture was quenched to room temperature in a desicator, ground in a ball mill (Retsch PM4, Brinkman, NY) and sieved to obtain TTCP powder with a median particle size of 5 μm. The commercial DCPA powder was ground for 24 h in the ball mill in 95% ethanol and sieved to obtain a median particle size of approximately 1 μm. Then the TTCP and DCPA powders at 1:3 molar ratio were thoroughly mixed in a micromill (Bel-Alert Products, Pequannock, NJ) to form the CPC powder . The microbeads were incorporated into CPC at 50% by volume. Degradable suture fibers (Ethicon, Somerville, NJ) were cut into 3 mm filaments and added into CPC at 10% by volume for mechanical reinforcement. The CPC liquid was 0.2 M Na 2 HPO 4 . A powder: liquid ratio of 2:1 was used to form a flowable CPC paste.
2.8
In vivo double cranial bone defects in rats
Double cranial defects of 5 mm each were created in 8-w-old male athymic nude rats (Hsd:RH-Fox1 mu , 200–250 g, Harlan, Indianapolis, IN) following a protocol approved by the University of Maryland Baltimore (IACUC # 0909014) and NIH animal-care guidelines. Briefly, under general anesthesia, two full-thickness 5 mm defects were made in the calvarium under continuous saline irrigation . CPC paste was mixed at the time of implantation and set in situ to fill the bone defects. Four groups were tested: (1) CPC-microbeads without cells (CPC control); (2) CPC-microbeads-iPSMSCs (CPC-iPSMSCs); (3) CPC-microbeads-OS-iPSMSCs (CPC-OS-iPSMSCs); (4) CPC-microbeads-BMP2-iPSMSCs (CPC-BMP2-iPSMSCs). Grafts were harvested after 12 weeks ( n = 5).
2.9
Histomorphometric analyses
Specimens were decalcified and embedded in paraffin. The central part of the implant and defect was cut into 5 μm-thick sections and stained with hematoxylin and eosin (HE) and Masson’s Trichrome (MT). New bone area, residual material area and total defect area was measured within the boundaries of defects in each section by Image Pro Plus Software (Media Cybernetics, Carlsbad, CA). New bone area fraction (NBAF) was calculated as the new bone area divided by total defect area ( n = 5). CPC scaffold residual material area fraction (RMAF) was calculated as the residual scaffold material area divided by total defect area ( n = 5).
2.10
Identification of encapsulated cells by immunohistochemistry (IHC)
Human origin of engineered bone constructs following in vivo implantation was detected using mouse monoclonal anti-human nuclei antibodies (Millipore). Tissue sections were deparaffinized with xylene, and rehydrated with a graded series of ethanol washes. The epitopes were recovered by incubation in citrate buffer at 70 °C for 40 min, and the endogenous peroxidase activity was blocked with 3% H 2 O 2 . The slides were then blocked with 1% BSA for 30 min to suppress nonspecific staining and stained with primary antibodies (1:50) overnight in a humidified environment. The specimens were subsequently incubated with secondary antibody against mouse IgG (1:500) for 30 min at 37 °C. Incubation was followed by streptavidin-HRP and diaminobenzidine (DAB) substrate, and counterstaining with hematoxylin solution. Negative controls were performed following the same procedures but without primary antibody incubation.
2.11
Statistical analyses
Statistical analyses were performed using Statistical Package for the Social Sciences (SPSS 19.0, Chicago, IL). All data were expressed as the mean value ± standard deviation (SD). All in vitro tests were independently repeated three times, with at least triplicate cultures for each condition. Statistical significance was analyzed by using the one-way analyses of variance (ANOVA) and Student–Newman–Keuls test. A confidence level of 95% ( p < 0.05) was considered significant.
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