The objective of this study was to investigate if the in vitro pre-culture period in osteogenic media of rat mesenchymal stem cells (MSCs), influences their ability to regenerate bone when implanted in a critical size cranial defect. MSCs were harvested from the bone marrow of 6-8 weeks old male Fisher rats and expanded in vitro in osteogenic media for different time periods (4, 10, and 16 days) in tissue culture plates (TCP), seeded on sintered titanium fiber meshes without the extracellular matrix (ECM) generated in vitro, and implanted in the rat cranium after 12 h. Thirty two adult Fisher rats received the implants, divided in four groups. Three groups were implanted with cells cultured for 4, 10, or 16 days in osteogenic media and at that time their alkaline phosphatase activity and mineral deposition denoted that they were at different stages of their osteoblastic maturation (undifferentiated MSC, committed, and mature Osteoblasts, respectively). MSCs cultured without osteogenic media for 6 days were used as controls. The constructs were retrieved 4 weeks later and processed for histomorphometric analysis. Implants seeded with cells that have been cultured with osteogenic media for only 4 days revealed the highest bone formation. The lowest bone formation was obtained with the implants seeded with MSCs cultured for 16 days in the presence of osteogenic media. The results of this study suggested that the in vitro pre-culture period of MSCs is a critical factor for their ability to regenerate bone when implanted to an orthotopic site.

Engineered bone grafts have been generated in static and dynamic systems by seeding and culturing osteoblastic cells on 3-D scaffolds. Seeding determines initial cellularity and cell spatial distribution throughout the scaffold, and affects cell-matrix interactions. Static seeding often yields low seeding efficiencies and poor cell distributions; thus creating a need for techniques that can improve these parameters. We have evaluated the effect of oscillating flow perfusion on seeding efficiency and spatial distribution of MC3T3-E1 pre-osteoblastic cells in fibrous polystyrene matrices (20, 35 and 50-microm fibers) and foams prepared by salt leaching, using as controls statically seeded scaffolds. An additional control was investigated where static seeding was followed by unidirectional perfusion. Oscillating perfusion resulted in the most efficient technique by yielding higher seeding efficiencies, more homogeneous distribution and stronger cell-matrix interactions. Cell surface density increased with inoculation cell number and then reached a maximum, but significant detachment occurred at greater flow rates. Oxygen plasma treatment of the fibers greatly improved seeding efficiency. Having similar porosity and dimensions, fibrous matrices yielded higher cell surface densities than foams. Fluorescence microscopy and histological analyses in polystyrene and PLLA scaffolds demonstrated that perfusion seeding produced more homogeneous cell distribution, with fibrous matrices presenting greater uniformity than the foams.

Arg-Gly-Asp (RGD) has been widely utilized to increase cell adhesion to three-dimensional scaffolds for tissue engineering. However, cell seeding on these scaffolds has only been carried out statically, which yields low cell seeding efficiencies. We have characterized, for the first time, the seeding of rat mesenchymal stem cells on RGD-modified poly(L-lactic acid) (PLLA) foams using oscillatory flow perfusion. The incorporation of RGD on the PLLA foams improves scaffold cellularity in a dose-dependent manner under oscillatory flow perfusion seeding. When compared to static seeding, oscillatory flow perfusion is the most efficient seeding technique. Cell detachment studies show that cell adhesion is dependent on the applied flow rate, and that cell attachment is strengthened at higher levels of RGD modification.

The present study combines chemical and mechanical stimuli to modulate the osteogenic differentiation of mesenchymal stem cells (MSCs). Arg-Gly-Asp (RGD) peptides incorporated into biomaterials have been shown to upregulate MSC osteoblastic differentiation. However, these effects have been assessed under static culture conditions, while it has been reported that flow perfusion also has an enhancing effect on MSC osteoblastic differentiation. It is clear that there is a need to combine RGD modification of biomaterials with mechanical stimulation of MSCs via flow perfusion and evaluate its effects on MSC differentiation down the osteogenic lineage. In this study, the effect of different levels of RGD modification of poly(L-lactic acid) scaffolds on MSC osteogenesis was evaluated under conditions of flow perfusion. It was found that there is a synergistic enhancement of different osteogenic markers, due to the combination of flow perfusion and RGD surface modification when compared to their individual effects. Furthermore, under conditions of flow perfusion, there is an RGD surface concentration optimal for differentiation, and it is flow rate-dependent. This report underlines the significance of incorporating combined biomimesis via biochemical and mechanical microenvironments that modulate in vivo cell behaviour and tissue function for more efficient tissue-engineering strategies.

Poly(L-lactic acid) (PLLA) is widely used in tissue-engineering applications because of its degradation characteristics and mechanical properties, but it possesses an inert nature, affecting cell-matrix interactions. It is desirable to modify the surface of PLLA to create biomimetic scaffolds that will enhance tissue regeneration. We prepared a functionally flexible, biomimetic scaffold by derivatizing the surface of PLLA foams into primary amines, activated pyridylthiols, or sulfhydryl groups, allowing a wide variety of modifications. Poly(L-lysine) (polyK) was physically entrapped uniformly throughout the scaffold surface and in a controllable fashion by soaking the foams in an acetone-water mixture and later in a polyK solution in dimethylsulfoxide. Arginine-glycine-aspartic acid-cysteine (RGDC) adhesion peptide was linked to the polyK via creating disulfide bonds introduced through the use of the linker N-succinimidyl-3-(2-pyridylthiol)-propionate. Presence of RGDC on the surface of PLLA 2-dimensional (2-D) disks and 3-D scaffolds increased cell surface area and the number of adherent mesenchymal stem cells. We have proposed a methodology for creating biomimetic scaffolds that is easy to execute, flexible, and nondestructive.

UV crosslinking of CH is easy to apply and has potential for future metallic implant surface modifications. Due to its nature as a hydrogel, the coating could be used for further studies in the encapsulation of bioactive molecules to improve osteogenic potential at the tissue-implant interface.

The fresh or cryopreserved human umbilical cord (HUC) and its byproducts, such as cells and extracts, have different uses in tissue regeneration. Defining what HUC byproduct is more effective in a particular application is a challenge. Furthermore, the methods of isolation, culture and preservation, may affect cell viability and regenerative properties. In this article, we review the HUC and its byproducts' applications in research and clinical practice. We present our results of successful use of HUC as a patch to treat gastroschisis and its potential to be applied in other conditions. Our in vitro results show an increase in proliferation and migration of human fibroblasts by using an acellular HUC extract. Our goal is to promote standardization of procedures and point out that applications of HUC and its byproducts, as well as the resulting advances in regenerative medicine, will depend on rigorous quality control and on more research in this area.

Milk thistle contains compounds that display hepatoxic protection properties. We examined the batch extraction of silymarin compounds from milk thistle seed meal in 50, 70, 85, and 100 degrees C water as a function of time. After 210 min of extraction at 100 degrees C, the yield of taxifolin was 1.2 mg/g of seed, a 6.2-fold increase over the results obtained in a Soxhlet extraction with ethanol on pretreated (defatted) seeds. Similarly, the yield of silychristin was 5.0 mg/g of seed, a 3.8-fold increase. The yields of silybinin A and silybinin B were 1.8 and 3.3 mg/g of seed, respectively, or roughly 30% of the Soxhlet yield. The ratios of the extracted compounds, and particularly the ratios at long extraction times, showed that the more polar compounds (taxifolin and silychristin) were preferentially extracted at 85 degrees C, while the less polar silybinin was favored at 100 degrees C.