In the field of bone tissue engineering and regenerative medicine, generating patient-specific stem cells has been a long-standing goal. Human bone marrow mesenchymal stem cells(hBMSCs) are the current gold-standard cell source for many tissue-engineering therapies and have been successfully applied in clinics. However, hBMSCs are hampered by an invasive harvesting procedure, limited availability, and loss of potency in
seniors or patients with certain diseases and disorders. Human umbilical cord MSCs (hUCMSCs) are capable of differentiating into mesenchymal lineages. They are easily and abundantly available, with robust proliferation and self-renewal capability due to their origin in neonatal tissues . The main obstacle of hUCMSCs is immunogenic concerns when used heterologously. For autologous applications, the umbilical cord has to be properly cryopreserved from childbirth for an extended period of time.
The harvested hiPSC–MSCs, hUCMSCs and hBMSCs were used in the following experiments.
Cells were seeded onto the RGD-biofunctionalized CPC disks in 24-well plates at a density of
3 ×105 cells/well . On the second day, the medium was changed into osteogenic medium which
consisted of the MSC growth medium supplemented with 100 nM dexamethasone, 10 mM
?-glycerophosphate, 0.05 mM ascorbic acid, and 10 nM 1a,25-dihydroxyvitamin (Sigma) .
At 1, 7 and 14 days, live/dead staining (Molecular Probes, Eugene, OR) was used to test cell
viability on CPC. Disks were washed with PBS and incubated with 4 mM ethidium homodimer-1
(EthD-1) and 2 mM calcein-AM in PBS for 20 min. The disks were examined using
epifluorescence microscopy (Eclipse TE2000-S, Nikon, Melville, NY). The percentage of live
cells P and the live cell density D were calculated as previously described . P = number of live
cells/(number of live cells + number of dead cells). D = number of live cells in the image/the
image area. Two randomly-chosen images for each sample were analyzed with three disks
per condition, yielding six images per group at each time point (technical replicates n = 6).
The test was independently repeated three times (biological replicates = 3) on three
different days by seeding new batches of cells on new batches of CPC disks.
Fig. 1. Cell viability when seeded on CPC scaffold. Live/dead staining of cells at 1 day and 14 days are shown in (A–F). In all three groups, live cells were abundant, and dead cells were few. Percentage of live cells on CPC was around 90% (G). All groups exhibited increasing live cell density (H). Bars with dissimilar letters indicate signi?cantly different values (p < 0.05). Each value is mean ± sd (technical replicates n = 6). The test was independently repeated three times (biological replicates = 3).
For osteogenic differentiation, at 1, 7 and 14 days, RNA was extracted from six cell-seeded disks per group per time point. Two disks were pooled together as an individual RNA sample to have suf?cient amount of RNA (technical replicates n = 3). The test was independently repeated
three times (biological replicates = 3) on three different days by seeding new batches of cells on new batches of CPC disks. The total cellular RNA was extracted with TRIzol reagent (Invitrogen) and PureLink RNA Mini Kit (Invitrogen), and then reverse-transcribed into cDNA by a High-capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). TaqMan gene expression kits were used to quantify targeted genes on human alkaline phosphatase (ALP, Hs00758162_m1), Runt-re-lated transcription factor (Runx2, Hs00231692_m1), collagen type I (COL1, Hs00164004), osteocalcin (OC, Hs00609452_g1), and glyc-eraldehyde 3-phosphate dehydrogenase (GAPDH, Hs99999905).Relative expression was evaluated using the 2DDCt method and normalized by the Ct of the housekeeping gene GAPDH. The Ct val-ue of hiPSC–MSCs, hUCMSCs, hBMSCs cultured on CPC scaffolds in growth medium for 1 day served as the calibrator .
For cell mineralization, at 1, 7 and 14 days, disks were ?xed with 10% formaldehyde and stained with Alizarin Red S (ARS, Mil-lipore, Billerica, MA) for 1 h. The disks were then rinsed by deion-ized water four times to visualize the presence of calci?ed deposition by the cells (technical replicates n =6) . An Osteo-genesis Quantitation Kit (Millipore, ECM 815) was used to extract the stained minerals and measure the ARS concentration, following the manufacturer’s instructions (n = 6). Control CPC scaffolds with the same compositions, but without cells, were measured at the same time periods; they were subjected to the same culture medi-um and incubation conditions as the cell-seeded disks (n = 6). The control’s ARS concentration was subtracted from that of the cell-seeded scaffolds to yield the net mineral concentration synthesized by the cells . The test was independently repeated three times (biological replicates = 3).
Fig. 2. Osteogenic differentiation of cells on CPC. RT-PCR results for ALP (A), Runx2 (B), COL1 (C), and OC (D). Each value is mean ± sd (technical replicates n = 3). The test was independently repeated three times (biological replicates = 3). Osteogenic markers were upregulated in all groups. OC, the late osteogenic marker, decreased in hiPSC-MSCs group, indicating that hiPSC–MSCs did not progress completely to mature osteoblasts in the in vitro induction. Mineral synthesis by cells was detected by Alizarin Red staining (E). The bone mineral matrix became denser and darker red with increasing time. Cell-synthesized mineral concentration was measured by the osteogenesis assay (F)(technical replicates n = 6 for mineralization). The test was independently repeated three times (biological replicates = 3). Bars with dissimilar letters indicate signi?cantly different values (p < 0.05).
Critical-sized cranial defects were created in male athymic nude rats (Hsd:RH-Fox1mu, 8 weeks old, weighing 200–250 g, Harlan, Indianapolis, IN) in accordance with the protocol approved by the University of Maryland (IACUC # 0909014) and NIH guidelines. Under general anesthesia of 75 mg/kg body weight of ketamine and 10 mg/kg of xylazine, a skin incision was made on the midline of cranium. The periosteum was ablated, and a full-thickness stan-dardized trephine defect, 8 mm in diameter, was made in the cal-varium under continuous saline buffer irrigation. Cell-seeded CPC scaffolds were maintained in osteogenic media for 14 days before implantation. Cell-free CPC was used as control. Rats were random-ly divided into four groups for: hiPSC–MSC–CPC scaffold, hUCMSC–CPC scaffold, hBMSC–CPC scaffold, and CPC control without cells, with six rats per group (technical replicates n = 6). The grafts were harvested at 12 weeks (w) and ?xed in 10% formalin.
Samples harvested at 12 weeks were scanned with micro-CT (lCT40, Scanco Medical, Bassersdorf, Switzerland). The parameters were set at a resolution of 18 lm, I = 114 lA, E = 70 kVp, with inte-gration time of 300 ms. Specimens were decalci?ed and embedded in paraf?n. The cen-tral region of the implant and defect was cut into 5 lm thick sec-tions and stained with hematoxylin and eosin (H&E) and Masson’s Trichrome. Six samples per group were evaluated and scored with a quan-titative grading scale (Table 1) . Samples were assessed for:(1) hard tissue response at the bone-scaffold interface; (2) bone bridging at the dura side of the defect; and (3) bone formation within the scaffold pores.
Fig. 3. Representative sagital micro-CT images of hiPSC–MSC–CPC (A), hUCMSCs (C), hBMSCs (D), and CPC control (E). Pro?le lines are shown for hiPSC–MSC–CPC (B), and CPC control (F). New bone is marked by arrows. A pro?le line was draw from the peripheral host bone, across the defect area to the other side of the defect boundary. The gray values on pro?le line were demonstrated in a curve diagram. Cell-seeded scaffolds exhibited higher signals (high intensity areas) than peripheral host bone. In contrast, CPC control had no such distinguishable differences in bone density, indicating less new bone.
Fig. 4. Representative H&E images. New bone areas were stained in pink red (arrows). The white area was due to slight detachment of the tissue or decalci?cation of CPC. The dark, dark purple and light purple areas were residual CPC. The periosteal side is on the top side, while the dura side is at the bottom. New bone (arrows) was mainly found on the dura side, and some new bone was deposited within the macropores of CPC. There was more new bone in cell-seeded groups than cell-free control.
Fig. 5. Representative Masson’s trichrome staining images. In cell-seeded groups, mineralized (deep blue areas) and nonmineralized osteoid (orange-red) mainly deposited on the dura side, and some new bone was formed within the pores of the scaffold. CPC control group had much less new bone.