Proliferation of seeded cells in cell-scaffold constructs was detected by cell counting kit-8 (A) and osteoblastic differentiation of seeded cells in cell-scaffold constructs was evaluated by ALP activities (B).

<p>The number of cells was increased with culture time except group C. The dynamic culture (groups A and B) showed an obvious ability of promoting proliferation of cells. The ALP activities in all groups increased from day 2 to day 14 (B). The ALP activities in groups A, B, D were statistically higher than that in groups C(p<0.05) from day 4 to day 14. indicates a statistically higher value compared with group C(p<0.05).</p

The culture and characterization of hMSCs.

<p>The hMSCs formed calcium nodus after 12 day culture (A). The hMSCs (200×) stained immunohistochemically positive for ALP (B), osteocalcin (E) and collagen type I (H) compared to non-induced cells (C, F, and I). The ALP activity (D) of hMSCs and osteocalcin concentration (G) in the culture medium were significant higher in induced group than that in control group (non-induced cells). *p < 0. 01, compared with control group.</p

Implant groups for subcutaneous implantation.

<p>Implant groups for subcutaneous implantation.</p

Summary of in vitro preparation of cell-scaffold constructs.

*<p>: seeding efficiency in group C was lower than that in other groups (<i>p<</i>0.05);</p>#<p>: seeding efficiency in group A was lower than that in group B and D (<i>p<</i>0.05).</p

Phase-contrast photomicrographs (×100) of cell-scaffold constructs after in vitro culture for 12 d; (A) group A (dynamic seeding and dynamic culture), (B) group B (hydrogel-assisted seeding and static flask culture, (C) group C (static seeding and static flask culture, control group), and (D) group D (hydrogel-assisted seeding and dynamic culture).

<p>Bar lengths are 100 um.</p

Photomicrographs (×100, methyl violet staining) of cell-scaffold constructs after in vitro culture for 12 d.

<p>The number of attached cells and density of extracellular matrix (ECM) fibers in the interior of the scaffold are obvious different among four groups, with group B (B) > group D (D) > group A (A) > group C (C). Bar lengths are 100 um.</p

Wet weight and bone mineral density of implants after subcutaneous implantation in nude mice.

<p>At 12 weeks postoperative, implant in group II showed higher wet weight (A) and bone mineral density (B) than that in other groups(p<0.05). *indicates a statistically significantly lower value compared with other implants; # indicates a statistically higher value compared with other implants.</p

Nude mice subcutaneous implantation model for the evaluation of osteogenic activity; (A) a photograph showing a nude mouse with four implants; (B) a radiograph 4 weeks after implantation; (C) a radiograph 8 weeks after implantation; (D) a radiograph 12 weeks after implantation.

<p>The radiographic densities of the implants increased from week 4 to week 12. The osteogenesis of implants was not clear at weeks 4 and 8 postoperative. It was not until 12 weeks postoperative that the imagings of implants in the radiographs were clearly observed. At week 12, implant II clearly showed increased density indicating calcification.</p

Scanning electron micrographs of cell-scaffold constructs after in vitro culture for 12 days.

<p>The attached cells and extracellular matrix (ECM) fibers presented on the scaffolds in group B (B) and group D (D) are significantly outnumber those in group A (A) as well as group C (C).Bar lengths are 100 um. The black arrows indicate cells and the blue arrows indicate ECM fibers.</p

Umbilical Cord Wharton’s Jelly Repeated Culture System: A New Device and Method for Obtaining Abundant Mesenchymal Stem Cells for Bone Tissue Engineering

<div><p>To date, various types of cells for seeding regenerative scaffolds have been used for bone tissue engineering. Among seed cells, the mesenchymal stem cells derived from human umbilical cord Wharton’s jelly (hUCMSCs) represent a promising candidate and hold potential for bone tissue engineering due to the the lack of ethical controversies, accessibility, sourced by non-invasive procedures for donors, a reduced risk of contamination, osteogenic differentiation capacities, and higher immunomodulatory capacity. However, the current culture methods are somewhat complicated and inefficient and often fail to make the best use of the umbilical cord (UC) tissues. Moreover, these culture processes cannot be performed on a large scale and under strict quality control. As a result, only a small quantity of cells can be harvested using the current culture methods. To solve these problems, we designed and evaluated an UC Wharton’s jelly repeated culture device. Using this device, hUCMSCs were obtained from the repeated cultures and their quantities and biological characteristics were compared. We found that using our culture device, which retained all tissue blocks on the bottom of the dish, the total number of obtained cells increased 15–20 times, and the time required for the primary passage was reduced. Moreover, cells harvested from the repeated cultures exhibited no significant difference in their immunophenotype, potential for multilineage differentiation, or proliferative, osteoinductive capacities, and final osteogenesis. The application of the repeated culture frame (RCF) not only made full use of the Wharton’s jelly but also simplified and specified the culture process, and thus, the culture efficiency was significantly improved. In summary, abundant hUCMSCs of dependable quality can be acquired using the RCF.</p></div