Recapitulating bone development events in a customised bioreactor through interplay of oxygen tension, medium pH, and systematic differentiation approaches.

Bone development and homeostasis are intricate processes that require co-existence and dynamic interactions among multiple cell types. However, controlled dynamic niches that derive and support stable propagation of these cells from single stem cell source is not sustainable in conventional culturing vessels. In bioreactor cultures that support dynamic niches, the limited source and stability of growth factors are often a major limiting factor for long-term in vitro cultures. Hence, alternative growth factor-free differentiation approaches are designed and their efficacy to achieve different osteochondral cell types is investigated. Briefly, a dynamic niche is achieved by varying medium pH, oxygen tension (pO2 ) distribution in bioreactor, initiating chondrogenic differentiation with chondroitin sulphate A (CSA), and implementing systematic differentiation regimes. In this study, we demonstrated that CSA is a potent chondrogenic inducer, specifically in combination with acidic medium and low pO2 . Further, endochondral ossification is recapitulated through a systematic chondrogenic-osteogenic (ch-os) differentiation regime, and multiple osteochondral cell types are derived. Chondrogenic hypertrophy was also enhanced specifically in high pO2 regions. Consequently, mineralised constructs with higher structural integrity, volume, and tailored dimensions are achieved. In contrast, a continuous osteogenic differentiation regime (os-os) has derived compact and dense constructs, whereas a continuous chondrogenic differentiation regime (ch-ch) has attenuated construct mineralisation and impaired development. In conclusion, a growth factor-free differentiation approach is achieved through interplay of pO2 , medium pH, and systematic differentiation regimes. The controlled dynamic niches have recapitulated endochondral ossification and can potentially be exploited to derive larger bone constructs with near physiological properties.

Synergistic effect of bimodal pore distribution and artificial extracellular matrices in polymeric scaffolds on osteogenic differentiation of human mesenchymal stem cells.

The main objective of this study was to enhance the biological performance of resorbable polymeric scaffolds for bone tissue engineering. Specifically, we focused on both microstructure and surface modification of the scaffolds to augment adhesion, proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSC). Moreover, a new cell seeding method assuring 90% seeding efficiency on the scaffolds was developed. Poly(l­lactide­co­glycolide) (PLGA) scaffolds with monomodal and bimodal pore distribution were produced by solvent casting/phase separation followed by porogen leaching and modified with artificial extracellular matrices (aECM) consisting of collagen type I and high sulphated hyaluronan (sHya). The application of two porogens resulted in bimodal pore distribution within the PLGA scaffolds as shown by scanning electron microscopy and microcomputer tomography. Two types of pores with diameters 400-600 µm and 2-20 µm were obtained. The scaffolds were successfully coated with a homogenous layer of aECM as shown by Sirius red and toluidine blue staining. In vitro study showed that presence of bimodal pore distribution in combination with collagen/sHya did not significantly influence hMSC proliferation and early osteogenic differentiation compared to scaffolds with monomodal pore distribution. However, it enhanced mineralization as well as the expression of Runt-related transcription factor 2, osteopontin and bone sialoprotein II. As a result PLGA scaffolds with bimodal pore distribution modified with collagen/sHya can be considered as prospective material promoting bone regeneration.

17-DMAG regulates p21 expression to induce chondrogenesis in vitro and in vivo.

Cartilage degeneration after injury affects a significant percentage of the population, including those that will go on to develop osteoarthritis (OA). Like humans, most mammals, including mice, are incapable of regenerating injured cartilage. Interestingly, it has previously been shown that p21 (Cdkn1a) knockout (p21-/-) mice demonstrate auricular (ear) cartilage regeneration. However, the loss of p21 expression is highly correlated with the development of numerous types of cancer and autoimmune diseases, limiting the therapeutic translation of these findings. Therefore, in this study, we employed a screening approach to identify an inhibitor (17-DMAG) that negatively regulates the expression of p21. We also validated that this compound can induce chondrogenesis in vitro (in adult mesenchymal stem cells) and in vivo (auricular cartilage injury model). Furthermore, our results suggest that 17-DMAG can induce the proliferation of terminally differentiated chondrocytes (in vitro and in vivo), while maintaining their chondrogenic phenotype. This study provides new insights into the regulation of chondrogenesis that might ultimately lead to new therapies for cartilage injury and/or OA.

p21-/- mice exhibit enhanced bone regeneration after injury.

BACKGROUND: p21(WAF1/CIP1/SDI1), a cyclin dependent kinase inhibitor has been shown to influence cell proliferation, differentiation and apoptosis; but more recently, p21 has been implicated in tissue repair. Studies on p21(-/-) knockout mice have demonstrated results that vary from complete regeneration and healing of tissue to attenuated healing. There have however been no studies that have evaluated the role of p21 inhibition in bone healing and remodeling. METHODS: The current study employs a burr-hole fracture model to investigate bone regeneration subsequent to an injury in a p21-/- mouse model. p21-/- and C57BL/6 mice were subjected to a burr-hole fracture on their proximal tibia, and their bony parameters were measured over 4 weeks via in vivo µCT scanning. RESULTS: p21-/- mice present with enhanced healing from week 1 through week 4. Differences in bone formation and resorption potential between the two mouse models are assessed via quantitative and functional assays. While the µCT analysis indicates that p21-/- mice have enhanced bone healing capabilities, it appears that the differences observed may not be due to the function of osteoblasts or osteoclasts. Furthermore, no differences were observed in the differentiation of progenitor cells (mesenchymal or monocytic) into osteoblasts or osteoclasts respectively. CONCLUSIONS: Therefore, it remains unknown how p21 is regulating enhanced fracture repair and further studies are required to determine which cell type(s) are responsible for this regenerative phenotype.

Developing a Customized Perfusion Bioreactor Prototype with Controlled Positional Variability in Oxygen Partial Pressure for Bone and Cartilage Tissue Engineering.

Skeletal development is a multistep process that involves the complex interplay of multiple cell types at different stages of development. Besides biochemical and physical cues, oxygen tension also plays a pivotal role in influencing cell fate during skeletal development. At physiological conditions, bone cells generally reside in a relatively oxygenated environment whereas chondrocytes reside in a hypoxic environment. However, it is technically challenging to achieve such defined, yet diverse oxygen distribution on traditional in vitro cultivation platforms. Instead, engineered osteochondral constructs are commonly cultivated in a homogeneous, stable environment. In this study, we describe a customized perfusion bioreactor having stable positional variability in oxygen tension at defined regions. Further, engineered collagen constructs were coaxed into adopting the shape and dimensions of defined cultivation platforms that were precasted in 1.5% agarose bedding. After cultivating murine embryonic stem cells that were embedded in collagen constructs for 50 days, mineralized constructs of specific dimensions and a stable structural integrity were achieved. The end-products, specifically constructs cultivated without chondroitin sulfate A (CSA), showed a significant increase in mechanical stiffness compared with their initial gel-like constructs. More importantly, the localization of osteochondral cell types was specific and corresponded to the oxygen tension gradient generated in the bioreactor. In addition, CSA in complementary with low oxygen tension was also found to be a potent inducer of chondrogenesis in this system. In summary, we have demonstrated a customized perfusion bioreactor prototype that is capable of generating a more dynamic, yet specific cultivation environment that could support propagation of multiple osteochondral lineages within a single engineered construct in vitro. Our system opens up new possibilities for in vitro research on human skeletal development.

Interplay of Substrate Conductivity, Cellular Microenvironment, and Pulsatile Electrical Stimulation toward Osteogenesis of Human Mesenchymal Stem Cells in Vitro.

The influences of physical stimuli such as surface elasticity, topography, and chemistry over mesenchymal stem cell proliferation and differentiation are well investigated. In this context, a fundamentally different approach was adopted, and we have demonstrated the interplay of inherent substrate conductivity, defined chemical composition of cellular microenvironment, and intermittent delivery of electric pulses to drive mesenchymal stem cell differentiation toward osteogenesis. For this, conducting polyaniline (PANI) substrates were coated with collagen type 1 (Coll) alone or in association with sulfated hyaluronan (sHya) to form artificial extracellular matrix (aECM), which mimics the native microenvironment of bone tissue. Further, bone marrow derived human mesenchymal stem cells (hMSCs) were cultured on these moderately conductive (10(-4)-10(-3) S/cm) aECM coated PANI substrates and exposed intermittently to pulsed electric field (PEF) generated through transformer-like coupling (TLC) approach over 28 days. On the basis of critical analysis over an array of end points, it was inferred that Coll/sHya coated PANI (PANI/Coll/sHya) substrates had enhanced proliferative capacity of hMSCs up to 28 days in culture, even in the absence of PEF stimulation. On the contrary, the adopted PEF stimulation protocol (7 ms rectangular pulses, 3.6 mV/cm, 10 Hz) is shown to enhance osteogenic differentiation potential of hMSCs. Additionally, PEF stimulated hMSCs had also displayed different morphological characteristics as their nonstimulated counterparts. Concomitantly, earlier onset of ALP activity was also observed on PANI/Coll/sHya substrates and resulted in more calcium deposition. Moreover, real-time polymerase chain reaction results indicated higher mRNA levels of alkaline phosphatase and osteocalcin, whereas the expression of other osteogenic markers such as Runt-related transcription factor 2, Col1A, and osteopontin exhibited a dynamic pattern similar to control cells that are cultured in osteogenic medium. Taken together, our experimental results illustrate the interplay of multiple parameters such as substrate conductivity, electric field stimulation, and aECM coating on the modulation of hMSC proliferation and differentiation in vitro.

Increased levels of p21((CIP1/WAF1)) correlate with decreased chondrogenic differentiation potential in synovial membrane progenitor cells.

Cartilage injuries are a major concern in the field of orthopedics. They occur following trauma, as well as from a variety of pathological conditions including Osteoarthritis (OA). Although cartilage does not exhibit robust endogenous repair, it has been demonstrated that modulating the activity of p21 can increase the regenerative abilities of cartilage in vitro and in vivo. Since the synovial membrane is abundant with mesenchymal progenitor cells (MPCs) capable of differentiating into cartilage both in vitro and in vivo, we examined if p21 expression levels varied between MPCs derived from normal vs. OA knee joints. Analysis of p21 at the mRNA and protein levels within normal and OA MPCs demonstrated differential levels of expression between these two groups, with OA MPCs having higher p21 expression levels. The higher levels of p21 in OA MPCs are also correlated with a decreased chondrogenic differentiation capacity and synovial inflammation, however, there was no evidence of senescence in the OA cells. The results of this study suggest that cell cycle regulation in MPCs may be altered in OA and that modulation of this pathway may have therapeutic potential once the mechanism by which this regulates stem/progenitor cells is better understood.

Healing properties of surface-coated polycaprolactone-co-lactide scaffolds: a pilot study in sheep.

The aim of this pilot study was to evaluate the bioactive, surface-coated polycaprolactone-co-lactide scaffolds as bone implants in a tibia critical size defect model. Polycaprolactone-co-lactide scaffolds were coated with collagen type I and chondroitin sulfate and 30 piled up polycaprolactone-co-lactide scaffolds were implanted into a 3 cm sheep tibia critical size defect for 3 or 12 months (n = 5 each). Bone healing was estimated by quantification of bone volume in the defects on computer tomography and microcomputer tomography scans, plain radiographs, biomechanical testing as well as by histological evaluations. New bone formation occurred at the proximal and distal ends of the tibia in both groups. The current pilot study revealed a mean new bone formation of 63% and 172% after 3 and 12 months, respectively. The bioactive, surface coated, highly porous three-dimensional polycaprolactone-co-lactide scaffold stack itself acted as a guide rail for new bone formation along and into the implant. These preliminary data are encouraging for future experiments with a larger group of animals.

Synergistic effect of defined artificial extracellular matrices and pulsed electric fields on osteogenic differentiation of human MSCs.

In vivo, bone formation is a complex, tightly regulated process, influenced by multiple biochemical and physical factors. To develop a vital bone tissue engineering construct, all of these individual components have to be considered and integrated to gain an in vivo-like stimulation of target cells. The purpose of the present studies was to investigate the synergistic role of defined biochemical and physical microenvironments with respect to osteogenic differentiation of human mesenchymal stem cells (MSCs). Biochemical microenvironments have been designed using artificial extracellular matrices (aECMs), containing collagen I (coll) and glycosaminoglycans (GAGs) like chondroitin sulfate (CS), or a high-sulfated hyaluronan derivative (sHya), formulated as coatings on three-dimensional poly(caprolactone-co-lactide) (PCL) scaffolds. As part of the physical microenvironment, cells were exposed to pulsed electric fields via transformer-like coupling (TC). Results showed that aECM containing sHya enhanced osteogenic differentiation represented by increases in ALP activity and gene-expression (RT-qPCR) of several bone-related proteins (RUNX-2, ALP, OPN). Electric field stimulation alone did not influence cell proliferation, but osteogenic differentiation was enhanced if osteogenic supplements were provided, showing synergistic effects by the combination of sHya and electric fields. These results will improve the understanding of bone regeneration processes and support the development of effective tissue engineered bone constructs.

Hydrostatic pressure stimulation of human mesenchymal stem cells seeded on collagen-based artificial extracellular matrices.

Human mesenchymal stem cells (hMSCs) from bone marrow are considered a promising cell source for bone tissue engineering applications because of their ability to differentiate into cells of the osteoblastic lineage. Mechanical stimulation is able to promote osteogenic differentiation of hMSC; however, the use of hydrostatic pressure (HP) has not been well studied. Artificial extracellular matrices containing collagen and chondroitin sulfate (CS) have promoted the expression of an osteoblastic phenotype by hMSCs. However, there has been little research into the combined effects of biochemical stimulation by matrices and simultaneous mechanical stimulation. In this study, artificial extracellular matrices generated from collagen and/or CS were coated onto polycaprolactone-co-lactide substrates, seeded with hMSCs and subjected to cyclic HP at various time points during 21 days after cell seeding to investigate the effects of biochemical, mechanical, and combined biochemical and mechanical stimulations. Cell differentiation was assessed by analyzing the expression of alkaline phosphatase (ALP) at the protein- and mRNA levels, as well as for calcium accumulation. The timing of HP stimulation affected hMSC proliferation and expression of ALP activity. HP stimulation after 6 days was most effective at promoting ALP activity. CS-containing matrices promoted the osteogenic differentiation of hMSCs. A combination of both CS-containing matrices and cyclic HP yields optimal effects on osteogenic differentiation of hMSCs on scaffolds compared with individual responses.