Acrylamide-based hydrogels with distinct osteogenic and chondrogenic differentiation potential.

Regeneration solutions for the osteochondral interface depth are limited, where multi-material implants have the potential to delaminate affecting the regeneration process and impacting the final integrity of tissue interface. Here we explore regionally mixed hydrogel networks, presenting distinct chemical features to determine their compatibility in supporting osteogenic or chondrogenic cell behaviour and differentiation. Poly(N-isopropylacrylamide) (pNIPAM) and poly(N-tert-butylacrylamide) (pNTBAM) hydrogels were assessed in terms of their chemical differences, mechanical strength, internal architecture, porosity and capacity to support cell viability, migration, and differentiation. pNTBAM polymerized with a Young's modulus of up to 371 ± 31 kPa compared to the more flexible pNIPAM, 16.5 ± 0.6 kPa. Viability testing revealed biocompatibility of both hydrogels with significantly increased cell numbers observed in pNTBAM (500 ± 95 viable cells/mm2) than in pNIPAM (60 ± 3 viable cells/mm2) (P ≤ 0.05). Mineralization determined through alkaline phosphatase (ALP) activity, calcium ion and annexin A2 markers of mineralization) and osteogenic behaviour (collagen I expression) were supported in both hydrogels, but to a greater extent in pNTBAM. pNTBAM supported significantly elevated levels of chondrogenic markers as evidenced by collagen II and glycosaminoglycan expression in comparison to little or no evidence in pNIPAM (P ≤ 0.05). In conclusion, structurally similar, chemically distinct, acrylamide hydrogels display variable capacities in supporting osteochondral cell behaviours. These systems demonstrate spatial control of cell interaction through simple changes in monomer chemistry. Fine control over chemical presentation during the fabrication of biomaterial implants could lead to greater efficacy and targeted regeneration of semi-complex tissues.

Vibrational spectroscopy in stem cell characterisation: is there a niche?

Vibrational spectroscopy using both infrared and Raman spectroscopies has been used in recent years with the aim to aid clinicians in disease diagnosis. More recently, these techniques have been applied to study stem cell differentiation and to determine stem cell presence in tissues. These studies have demonstrated the potential of these techniques in better characterising stem cell differentiation phenotypes with potential applications in tissue engineering strategies. However, before the translation of vibrational spectroscopy into clinical practice becomes a reality, several issues still need to be addressed. We describe here an overview of the work carried out so far and the problems that might be encountered when using vibrational spectroscopy.

Bone tissue formation from human embryonic stem cells in vivo.

Although the use of embryonic stem cells in the assisted repair of musculoskeletal tissues holds promise, a direct comparison of this cell source with adult marrow-derived stem cells has not been undertaken. Here we have compared the osteogenic differentiation potential of human embryonic stem cells (hESC) with human adult-derived stem cells in vivo. hESC lines H7, H9, the HEF-1 mesenchymal-like, telomerized H1 derivative, the human embryonic kidney epithelial cell line HEK293 (negative control), and adult human mesenchymal stem cells (hMSC) were either used untreated or treated with osteogenic factors for 4 days prior to injection into diffusion chambers and implantation into nude mice. After 11 weeks in vivo chambers were removed, frozen, and analyzed for evidence of bone, cartilage, and adipose tissue formation. All hESCs, when pretreated with osteogenic (OS) factors gave rise exclusively to bone in the chambers. In contrast, untreated hESCs (H9) formed both bone and cartilage in vivo. Untreated hMSCs did not give rise to bone, cartilage, or adipose tissue in vivo, while pretreatment with OS factors engendered both bone and adipose tissue. These data demonstrate that hESCs exposed to OS factors in vitro undergo directed differentiation toward the osteogenic lineage in vivo in a similar fashion to that produced by hMSCs. These findings support the potential future use of hESC-derived cells in regenerative medicine applications.

Fluorescence-activated single cell sorting of human embryonic stem cells.

Human embryonic stem cells (hESC) are the subject of intense investigation for use in regenerative medicine, in toxicity testing, and as models for the study of human development. Automated cell sorting will enhance the isolation of homogenous pools of differentiated hESCs both for basic studies and for therapeutic applications. Sorting could also be used to deplete undifferentiated, potentially tumourigenic cells. However, hESCs are sensitive to single cell disaggregation and recover poorly when plated at clonal density. Here we report a method for successful semi-automated single cell sorting of hESCs. This method utilizes an ES-specific promoter-transgene construct and automated FACS-based single cell sorting and plating. Clonal recovery in physiologic oxygen (2%) was increased fourfold over room oxygen (21%; p < 0.01). This automated protocol will help to realize proposed hESC strategies that are hampered by low throughput and poor yields.

Distinct telomere length regulation in premalignant cervical and endometrial lesions: implications for the roles of telomeres in uterine carcinogenesis.

Mouse models show that progressive shortening of telomeres with ageing causes chromosomal instability, which can lead to the initiation of cancer. However, it is unclear what roles telomere shortening plays in human carcinogenesis. The present study has investigated the involvement of telomere dynamics in uterine carcinogenesis. Using telomere-FISH (telo-FISH) assays, telomere lengths in premalignant and malignant cervical and endometrial lesions were measured and compared with chromosomal arm loss or gain. Telo-FISH signals were visualized with Cy3-labelled telomere-specific probes and presented as telomere intensity (TI). Early-stage cervical intraepithelial neoplasias (CINs), especially CIN2, had significantly shorter telomeres than corresponding normal squamous epithelia (p = 0.019), together with increased rates of chromosomal arm loss/gain (p < 0.001). Cervical cancers had relatively short telomeres, but they also showed greater heterogeneity than other sampled tissues, including those with long telomeres. In contrast, there was no significant difference between the telomere length of normal endometrium and of endometrial hyperplasia and endometrial cancer. There was no significant difference in the rate of chromosomal arm loss/gain between normal endometrium and endometrial hyperplasia. These findings suggest that progressive shortening of telomeres occurs in CIN, in association with chromosomal instability, which may play critical roles in cervical carcinogenesis. In contrast, endometrial hyperplasias have relatively stable telomeres without widespread chromosome alteration, implying that endometrial carcinogenesis involves mechanisms distinct from those of cervical carcinogenesis, possibly including microsatellite instability.

Genetic and functional analyses exclude mortality factor 4 (MORF4) as a keratinocyte senescence gene.

Approximately 50% of immortal human keratinocyte lines show loss of heterozygosity of chromosome region 4q33-q34, and the reintroduction of chromosome 4 into one such line, BICR 6, causes proliferation arrest and features of replicative senescence. Recently, a candidate gene, mortality factor 4 (MORF4), was identified in this region and sequenced in 21 immortal keratinocyte lines. There were no mutations or deletions, and two of the seven lines that showed loss of heterozygosity at 4q33-q34 were heterozygous for MORF4 itself. Furthermore, the transfer of a chromosomal segment containing the entire MORF4 gene did not mimic the senescence effect of chromosome 4 in BICR 6. These results suggest that the inactivation of MORF4 is not required for human keratinocyte immortality.