Inga Marijanović

Expression and Function of Dlx Genes in the Osteoblast Lineage

Our laboratory and others have shown that overexpression of Dlx5 stimulates osteoblast differentiation. Dlx5(-/-)/Dlx6(-/-) mice have more severe craniofacial and limb defects than Dlx5(-/-), some of which are potentially due to defects in osteoblast maturation. We wished to investigate the degree to which other Dlx genes compensate for the lack of Dlx5, thus allowing normal development of the majority of skeletal elements in Dlx5(-/-) mice. Dlx gene expression in cells from different stages of the osteoblast lineage isolated by FACS sorting showed that Dlx2, Dlx5 and Dlx6 are expressed most strongly in less mature osteoblasts, whereas Dlx3 is very highly expressed in differentiated osteoblasts and osteocytes. In situ hybridization and Northern blot analysis demonstrated the presence of endogenous Dlx3 mRNA within osteoblasts and osteocytes. Dlx3 strongly upregulates osteoblastic markers with a potency comparable to Dlx5. Cloned chick or mouse Dlx6 showed stimulatory effects on osteoblast differentiation. Our results suggest that Dlx2 and Dlx6 have the potential to stimulate osteoblastic differentiation and may compensate for the absence of Dlx5 to produce relatively normal osteoblastic differentiation in Dlx5 knockout mice, while Dlx3 may play a distinct role in late stage osteoblast differentiation and osteocyte function.

Overexpression of Dlx5 in chicken calvarial cells accelerates osteoblastic differentiation

Our laboratory and others have shown that a homeodomain protein binding site plays an important role in transcription of the Col1a1 gene in osteoblasts. This suggests that homeodomain proteins have an important role in osteoblast differentiation. We have investigated the role of Dlx5 in osteoblastic differentiation. In situ hybridization studies indicated that Dlx5 is expressed in chick calvarial osteoblasts (cCOB) in vivo. Northern blot analysis indicated that Dlx5 expression in cultured cCOBs is induced concurrently with osteoblastic markers. To study the effect of overexpression of Dlx5 on osteoblast differentiation, we infected primary osteoblast cultures from 15‐day‐old embryonal chicken calvaria with replication competent retroviral vectors [RCASBP(A)] expressing Dlx5 or control replication competent avian splice acceptor brianhightiter polymerase subtype A [RCASBP(A)]. Expression of Col1a1, osteopontin, alkaline phosphatase, and osteocalcin messenger RNA (mRNA) occurred sooner and at higher levels in cultures infected with RCASBP(A)DLX5 than in RCASBP(A)‐infected cultures. Mineralization of Dlx5‐expressing cultures was evident by days 12‐14, and RCAS‐infected control osteoblasts did not begin to mineralize until day 17. Dlx5 also stimulated osteoblastic differentiation of calvarial cells that do not normally undergo osteoblastic differentiation in vitro. Our results suggest that Dlx5 plays an important role in inducing calvarial osteoblast differentiation.

Human Mesenchymal Stem Cells Differentiation Regulated by Hydroxyapatite Content within Chitosan-Based Scaffolds under Perfusion Conditions

The extensive need for hard tissue substituent greatly motivates development of suitable allogeneic grafts for therapeutic recreation. Different calcium phosphate phases have been accepted as scaffold’s components with positive influence on osteoinduction and differentiation of human mesenchymal stem cells, in terms of their higher fraction within the graft. Nevertheless, the creation of unlimited nutrients diffusion through newly formed grafts is of great importance. The media flow accomplished by perfusion forces can provide physicochemical, and also, biomechanical stimuli for three-dimensional bone-construct growth. In the present study, the influence of a different scaffold’s composition on the human mesenchymal stem cells (hMSCs) differentiation performed in a U-CUP bioreactor under perfusion conditioning was investigated. The histological and immunohistochemical analysis of cultured bony tissues, and the evaluation of osteogenic genes’ expression indicate that the lower fraction of in situ formed hydroxyapatite in the range of 10–30% within chitosan scaffold could be preferable for bone-construct development.

Bioreactor-Based Bone Tissue Engineering

The aim of this chapter is to describe the main issues of bone tissue engineering. Bone transplants are widely used in orthopedic, plastic and reconstructive surgery. Current technologies like autologous and allogenic transplantation have several disadvantages making them relatively unsatisfactory, like donor site morbidity, chronic pain, and immunogenicity and risk hazard from infectious disease. Therefore, regenerative orthopedics seeks to establish a successful protocol for the healing of severe bone damage using engineered bone grafts. The optimization of protocols for bone graft production using autologous mesenchymal stem cells loaded on appropriate scaffolds, exposed to osteogenic inducers and mechanical force in bioreactor, should be able to solve the current limitations in managing bone injuries. We discuss mesenchymal stem cells as the most suitable cell type for bone tissue engineering. They can be isolated from a variety of mesenchymal tissues and can differentiate into osteoblasts when given appropriate mechanical support and osteoinductive signal. Mechanical support can be provided by different cell scaffolds based on natural or synthetic biomaterials, as well as combined composite materials. Three-dimensional support is enabled by bioreactor systems providing several advantages as mechanical loading, homogeneous distribution of cells and adequate nutrients/waste exchange. We also discuss the variety of osteoinductive signals that can be applied in bone tissue engineering. The near future of bone healing and regeneration is closely related to advances in tissue engineering. The optimization of protocols of bone graft production using autologous mesenchymal stem cells loaded on appropriate scaffolds, exposed to osteogenic inducers and mechanical force in bioreac‐ tor, should be able to solve the current limitations in managing bone injuries.

Association between Infection Aspects in Women with Squamous Intraepithelial Lesions and Peniscopic Findings in Their Male Sexual Partners

PURPOSE: To correlate colposcopy of women having squamous intraepithelial lesions (SIL) and peniscopic findings in their male sexual partners, to determine the prevalence of human papillomavirus (HPV) DNA in the male partners of women affected by SIL, and to assess the concordance of the viral group in the infected couple. PATIENTS AND METHODS: 27 females with different grade of SIL were examined by cytology, colposcopy and histology and their male sexual partners underwent clinical examination, peniscopy and biopsy. HPV testing was performed for all. RESULTS: 50 % of the sexual partners of women with low-grade squamous intraepithelial lesions (LSIL) were peniscopically negative. 36 % of the male partners of women with high-grade squamous intraepithelial lesions (HSIL) were peniscopically negative. Clinical lesions were observed in 50 and 9 % of the sexual partners of women with LSIL and HSIL, respectively, while subclinical lesions were found only in male partners of women with HSIL (45 %). Sexual partners of women with LSIL enrolled in our study were not HPV-positive. Male partners of patients with HSIL were HPV-positive in 63 % cases with 59 % being infected with high-risk viruses and 4 % with low-risk HPV viruses. The same genotypes were found in 19 % of couples, similarity in one genotype and difference in the other was found in 29.6 % of couples, while complete discordance was found in 48 % of couples. Highest concordance was achieved by high-risk types, especially type 16. CONCLUSION: Male sexual partners of women with SIL lesions have penile lesions determined by peniscopical examination that are mostly associated with the presence of high-risk HPV in women with HSIL. High-risk HPV prevalence is found among male partners with clinical and especially subclinical penile manifestations. Concordance of the viral group in infected pair needs to be more elucidated.