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A protocol for the extraction of DNA from ancient skeletal material was developed. Bone specimen samples (powder or slice), buffer, pretreatment, and extraction methodologies were compared to investigate the best conditions yielding the highest concentration of DNA. The degree of extract contamination by polymerase chain reaction (PCR) inhibitors was compared as well. Pretreatment was carried out using agitation in an incubator shaker and microwave digestion. Subsequently, DNA from bones was isolated by the classical organic phenol–chloroform extraction and silica-based spin columns. Decalcification buffer for total demineralization was required as well as lysis buffer for cell lysis to obtain DNA, whereas microwave-assisted digestion proved to be very rapid, with an incubation time of 2 min instead of 24 h at an incubator shaker without using lysis buffer. The correction of isolated DNA was detected using real-time PCR with melt curve analysis, which was 82.8 ± 0.2 °C for highly repetitive α-satellite gene region specific for human chromosome 17 (locus D17Z1). Consequently, microwave-based DNA digestion followed by silica column yielded a high-purity DNA with a concentration of 19.40 ng/μl and proved to be a superior alternative to the phenol–chloroform method, presenting an environmentally friendly and efficient technique for DNA extraction.

The effects of fabrication techniques were investigated for preparation of a chitosan based scaffold. A two-step process was used: fabrication of hydrogel which was subsequently processed either with supercritical CO2 (SC-CO2) or lyophilization. Various pressures from subcritical to supercritical (70, 160, 250 bar), temperatures (35, 45, 55 °C) and durations (2–3 h) were applied in order to elicit the optimum process parameters yielding the highest porosity which were determined as 250 bar, 45 °C, 2 h of processing at 5 g/min CO2 flow rate yielding a porosity of 87.03% which was similar to lyophilization (88.68%) achieved at 55 °C for 48 h. When osteosarcoma cells possessing cellular features of osteoblasts were seeded, SC-CO2 dried scaffold proved to be a more ideal support for cell attachment owing to the presence of both nano and micropores, thereby providing a more efficient and rapid alternative for tissue engineering applications.