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In this study, we present a method for assembling biofunctionalized paper into a multiform structured scaffold system for reliable tissue regeneration using an origami-based approach. The surface of a paper was conformally modified with a poly(styrene-co-maleic anhydride) layer via initiated chemical vapor deposition followed by the immobilization of poly-l-lysine (PLL) and deposition of Ca(2+). This procedure ensures the formation of alginate hydrogel on the paper due to Ca(2+) diffusion. Furthermore, strong adhesion of the alginate hydrogel on the paper onto the paper substrate was achieved due to an electrostatic interaction between the alginate and PLL. The developed scaffold system was versatile and allowed area-selective cell seeding. Also, the hydrogel-laden paper could be folded freely into 3D tissue-like structures using a simple origami-based method. The cylindrically constructed paper scaffold system with chondrocytes was applied into a three-ring defect trachea in rabbits. The transplanted engineered tissues replaced the native trachea without stenosis after 4 wks. As for the custom-built scaffold system, the hydrogel-laden paper system will provide a robust and facile method for the formation of tissues mimicking native tissue constructs.

This study was undertaken to determine whether periosteum from different bone sources in a donor results in the same formation of bone and cartilage. In this case, periosteum obtained from the cranium and mandible (examples of tissue supporting intramembranous ossification) and the radius and ilium (examples of tissues supporting endochondral ossification) of individual calves was used to produce tissue-engineered constructs that were implanted in nude mice and then retrieved after 10 and 20 weeks. Specimens were compared in terms of their osteogenic and chondrogenic potential by radiography, histology, and gene expression levels. By 10 weeks of implantation and more so by 20 weeks, constructs with cranial periosteum had developed to the greatest extent, followed in order by ilium, radius, and mandible periosteum. All constructs, particularly with cranial tissue although minimally with mandibular periosteum, had mineralized by 10 weeks on radiography and stained for proteoglycans with safranin-O red (cranial tissue most intensely and mandibular tissue least intensely). Gene expression of type I collagen, type II collagen, runx2, and bone sialoprotein (BSP) was detectable on QRT-PCR for all specimens at 10 and 20 weeks. By 20 weeks, the relative gene levels were: type I collagen, ilium >> radial ≧ cranial ≧ mandibular; type II collagen, radial > ilium > cranial ≧ mandibular; runx2, cranial >>> radial > mandibular ≧ ilium; and BSP, ilium ≧ radial > cranial > mandibular. These data demonstrate that the osteogenic and chondrogenic capacity of the various constructs is not identical and depends on the periosteal source regardless of intramembranous or endochondral ossification. Based on these results, cranial and mandibular periosteal tissues appear to enhance bone formation most and least prominently, respectively. The appropriate periosteal choice for bone and cartilage tissue engineering and regeneration should be a function of its immediate application as well as other factors besides growth rate.

Dentin matrix protein 1 (DMP1) has been shown to be critical for the formation of dentin and bone. However, the precise pathway by which DMP1 participates in dentinogenesis and osteogenesis remains to be clarified. DMP1 is present in the extracellular matrix of dentin and bone as processed NH2- and COOH-terminal fragments. The NH2-terminal fragment occurs as a proteoglycan, whereas the COOH-terminal fragment is highly phosphorylated. The differences in biochemical properties suggest that these fragments may have different tissue and cell distribution in association with distinct functions. In this study, we analyzed the distribution of the NH2- and COOH-terminal fragments of DMP1 in tooth, bone, osteocytes as well as MC3T3-E1 and HEK-293 cells. Immunohistochemical analyses were performed using antibodies specific to the NH2- or COOH-terminal region of DMP1. Clear differences in the distribution of these fragments were observed. In the teeth and bone, the NH2-terminal fragment was primarily located in the nonmineralized predentin and cartilage of the growth plate, while the COOH-terminal fragment accumulated in the mineralized zones. In osteocytes, the NH2-terminal fragment appeared more abundant along cell membrane and processes of osteocytes, while the COOH-terminal fragment was often found in the nuclei. This pattern of distribution in cellular compartments was further confirmed by analyses on MC3T3-E1 and HEK-293 cells transfected with a construct containing DMP1 cDNA. In these cell lines, the COOH-terminal fragment accumulated in cell nuclei, while the NH2-terminal fragment was in the cytosol. The different distribution of DMP1 fragments indicates that these DMP1 variants must perform distinct functions.

Coordinated activities of multiple mesenchymal cell types contribute to the development of the mammalian skeleton formed through endochondral ossification. Synthesis of a cartilage template by chondrocytes is an obligatory step for the generation of skeletal elements during endochondral ossification. Gene ablation studies have established that Runx2 is an essential transcription factor for bone formation and the differentiation of skeletal cells. However, global gene deletion has failed to discern the tissue- and cell type-specific roles of Runx2. We generated floxed mice to elucidate the Runx2 regulatory control distinctive to cartilage tissue during bone development. Exon 8 of the Runx2 gene was selectively deleted in developing chondrocytes by utilizing Col2a-Cre mice. Cell- and tissue-specific gene recombination was confirmed by β-gal activity in R26R mice. The chondrocyte-specific loss of Runx2 caused failure of endochondral ossification, impaired craniofacial development, dwarfism, and perinatal lethality. Radiographic imaging and histochemical approaches were used to characterize the skeletal phenotype. We conclude that regulatory control of Runx2 in chondrocytes is essential for endochondral ossification, and it is independent of the role of Runx2 in osteoblasts.

Dentin matrix protein 1 (DMP1) is an acidic protein that plays critical roles in osteogenesis and dentinogenesis. Protein chemistry studies have demonstrated that DMP1 primarily exists as processed NH2- and COOH-terminal fragments in the extracellular matrix of bone and dentin. Our earlier work showed that the substitution of Asp213 (a residue at a cleavage site) by Ala213 blocks the processing of mouse DMP1 in vitro. Recently, we generated transgenic mice expressing this mutant DMP1 (designated ‘D213A-DMP1’). By crossbreeding these transgenic mice with Dmp1-knockout (Dmp1-KO) mice, we obtained mice expressing the D213A-DMP1 transgene in the Dmp1-null background (named ‘Dmp1-KO/D213A-Tg’ mice). In this study, we analyzed the long bone, mandible, dentin, and cartilage of Dmp1-KO/D213A-Tg mice in comparison with wild-type, Dmp1-KO, and Dmp1-KO mice expressing the normal DMP1 transgene (Dmp1-KO/normal-Tg). Our results showed that D213A-DMP1 was barely cleaved in the dentin matrix of Dmp1-KO/D213A-Tg mice and the expression of D213A-DMP1 failed to rescue the developmental defects in Dmp1-null mice. Interestingly, enlarged growth plates and condylar cartilages were observed in Dmp1-KO/D213A-Tg mice, indicating a potential role of the full-length form of DMP1 in chondrogenesis.

Two O-hydroxylysyl glycosides, Hyl-Gal-Glc and Hyl-Gal, have been isolated from normal human urine and shown to be identical to two glycosides isolated from alkaline hydrolysates of collagen. A relatively sample and reproducible analytical procedure has been devised to measure the levels of these glycosides in human urine. By the use of this procedure it was shown that a normal diet has only a small effect on 24-hr urinary excretion levels of these glycosides indicating an endogenous origin. Urinary glycoside levels appear to be highest in children, roughly paralleling collagen turnover as indicated by urinary hydroxyproline levels. Collagen turnover equivalents calculated from urinary hydroxylysyl glycoside levels were found to be significantly larger than collagen turnover equivalents calculated from urinary hydroxyproline levels. This suggests that urinary glycosides are more quantitative indicators of collagen metabolism than urinary hydroxyproline. The ratio of Hyl-Gal-Glc to Hyl-Gal was measured in urines of diseased as well as normal individuals and a bimodal distribution was found. Alkaline hydrolysates of different human connective tissue collagens showed that only bone collagen, of the collagens examined, had a low ratio of Hyl-Gal-Glc to Hyl-Gal compared to human urine. Other collagens examined had higher ratios than found in human urine. On the basis of these results it is postulated that the bimodal distribution of glycoside ratios represents two populations of collagen turnover, the lower ratio population having a high bone collagen turnover, the lower ratio population having a high bone collagen turnover relative to the second population. Examination of the types of subjects making up the two populations supports this hypothesis. These data suggest that urinary O-hydroxylysyl glycoside excretion, in addition to providing a more quantitative estimate of collagen turnover than urinary hydroxyproline, may prove to be of value as a specific means of studying the metabolism of bone collagen.