Restoration of cellular function of mesenchymal stem cells from a hypophosphatasia patient.

Mesenchymal stem cells (MSCs) can differentiate into multiple cell lineages and are used for regenerative treatments for a variety of diseases. However, the patient's cells cannot be used to treat genetic diseases. Allogeneic cells can serve as an alternative but long-term survival is uncertain. Our experience of allo-transplantation to a patient with hypophosphatasia, which is caused by mutations of the tissue non-specific alkaline phosphatase (TNSALP) gene resulting in low serum alkaline phosphatase (ALP) activity and skeletal deformity, did not improve these clinical characteristics. Therefore, we sought to use autologous MSCs for the treatment of hypophosphatasia. MSCs derived from the patient's bone marrow had a similar profile when compared with well-reported MSCs. However, the MSCs had extremely low ALP activity and could not produce a mineralized bone matrix even under the osteogenic culture conditions. We therefore transduced a retroviral vector with TNSALP promoter-driven TNSALP gene in the MSCs. In the culture condition, the MSCs had about 7-fold higher ALP activity than did mock-transduced MSCs, and showed mineralization as well as bone-specific markers. Furthermore, the MSCs, but not mock-transduced MSCs, newly formed bone at the frequency of 50% in nude rats. Transplantation of the TNSALP-transduced autologous MSCs might become a new therapy for hypophosphatasia.

Spectrocolorimetric evaluation of human articular cartilage.

OBJECTIVE: The aim of this study was to investigate whether human articular cartilage can be quantitatively evaluated using a spectrocolorimeter. MATERIALS AND METHODS: Human articular cartilage specimens were analyzed using a spectrocolorimeter after macroscopic evaluation using the Outerbridge classification. The cartilage characteristics were examined, the L*, a*, b* colorimetric system, the spectral reflectance distribution and the yellow/red spectral reflectance percentage (Y/R SRP). Moreover, the results of the spectrocolorimetric evaluation were compared with the histological score described by Mankin et al. RESULTS: There were significant differences among the macroscopic four grades in the L*, a* and Y/R SRP values. The spectral reflectance distribution of grade 1 cartilage exhibited a gradual increase in the spectral reflectance ratio as the wavelength increased. The spectral reflectance curves of grades 2 to 4 cartilage had dips at a wavelength of around 580 nm. Across all the measured wavelengths, there were lower reflectance ratios with the progression of cartilage degeneration. Moreover, correlations were observed between the spectrocolorimetric values and Mankin score. A strong relationship existed between Mankin score and he Y/R SRP values. CONCLUSIONS: The present study is the first to clearly demonstrate the relationship between spectrocolorimetric evaluation and the degeneration of human articular cartilage. The spectrocolorimeter may be a new quantitative evaluation tool for articular cartilage with clinical potential.

Bone viability of amputated limbs treated with hypothermia: assessment by evaluation of mRNA levels.

We evaluated rat bone viability using a bone viability index (BVI). To evaluate hypothermic ischaemic bone injury, 21 amputated hind limbs of Fischer rats were preserved at hypothermia (4 degrees C) for 1, 3 and 6 hours. To evaluate hypothermic ischaemia/reperfusion injury, another 28 amputated limbs were transplanted to recipient rats after hypothermic ischaemia for 3 and 6 hours, respectively. Total RNA isolated from each tibia was fractionated by electrophoresis and hybridised with 32P-labelled cDNA of GAPDH, and the radioactivity of intact and degraded GAPDH mRNA measured. BVI was calculated as follows, BVI = [A / (A + B)] x 100, where A and B represent the radioactivities corresponding to intact and degraded GAPDH mRNA bands, respectively. In the hypothermic ischaemic insult group, BVIs were comparable to those of controls. However, in the 3-hour hypothermic ischaemia/reperfusion group, BVI was lower than that of the controls. Likewise, there was a significant difference between the 6-hour ischaemia/reperfusion group and controls. These results showed that bone viability decreased even after just a 3-hour hypothermic ischaemia/reperfusion.

In vivo osteogenic capability of cultured allogeneic bone in porous hydroxyapatite: immunosuppressive and osteogenic potential of FK506 in vivo.

Fischer or ACI rat marrow cells were obtained from femoral shafts and were cultured to confluence in Eagle's minimal essential medium (EMEM) supplemented with 15% fetal bovine serum. After trypsinization, the cells were subcultured on porous hydroxyapatite (HA; Interpore 500) blocks in the presence of beta-glycerophosphate and 10 nM dexamethasone (Dex). After 2 weeks of subculture, a mineralized bone matrix with osteogenic cells developed on the HA pore surfaces. ACI or Fischer cultured bone tissue/HA constructs were implanted subcutaneously into the backs of Fischer rats and the immunosuppressant FK506 was given to the rats for 4 weeks. Implants were harvested 4 weeks and 8 weeks after insertion. At 4 weeks, the ACI constructs (allografts) showed high levels of osteogenic parameters (alkaline phosphatase [ALP] activity and osteocalcin content) and bone formation was observed together with active osteoblasts without obvious accumulation of inflammatory cells. At 8 weeks, active osteoblasts and progressive bone formation were still observed, while osteogenic parameters remained high and osteocalcin messenger RNA (mRNA) was detected. Without FK506 administration, the allografts showed neither bone formation nor osteocalcin mRNA and there were only trace levels of the osteogenic parameters. In the case of Fischer constructs (isografts), extensive bone formation was detected and all the osteogenic parameters were higher with FK506 than without FK506 at both 4 weeks and 8 weeks. These results indicate that cultured bone tissue/HA constructs possess a high osteogenic potential, even as allografts, and that FK506 not only has an immunosuppressive action, but also promotes bone formation.

The effect of aging on bone formation in porous hydroxyapatite: biochemical and histological analysis.

The effect of aging on osteoblastic differentiation of marrow stromal stem cells was examined. Porous hydroxyapatite (HA) disks were soaked in cells suspensions of bone marrow cells from young (8 weeks) and old rats (60 weeks) and then implanted subcutaneously in syngeneic young and old rats. The bone marrow/HA composites were harvested 8 weeks later, and the contents of bone Gla protein (BGP) and alkaline phosphatase (ALP) activity in them were determined. Histologically, bone formation could be detected in all the composites in young recipient rats; however, some old bone marrow/HA composites in old recipients did not show bone formation and the bone volume in the young bone marrow/HA composites was greater than in the old bone marrow/HA composites. The ratios of ALP activities of young bone marrow/HA composites to old bone marrow/HA composites in young and old recipients were about five times and four times, respectively. The ratios of BGP contents of young bone marrow/HA to old bone marrow/HA composite in young and old recipients were about nine and eight times, respectively. The results suggest that the decreased bone formation observed in old bone marrow cells was due to a smaller population of stromal cells and/or decreased capacity of differentiation of stromal stem cells into osteogenic cells.

Osteogenesis associated with bone gla protein gene expression in diffusion chambers by bone marrow cells with demineralized bone matrix.

Diffusion chambers with rat bone marrow cells and demineralized bone matrix (DBM) were implanted subcutaneously to syngeneic 8-week-old rats and were harvested every week 3-7 weeks after implantation, and histochemical examination, determination of alkaline phosphatase activity, total calcium and phosphorus, the bone-specific vitamin K-dependent gla-containing protein (BGP) content, and detection of BGP mRNA relative to mineralization were performed. Alkaline phosphatase in diffusion chamber implants reached the highest activity at 4 weeks and then decreased. Calcium and phosphorus deposits occurred at 4 weeks after implantation and were followed by marked increases until 7 weeks, which was comparable to the accumulation of BGP. The BGP gene within the diffusion chambers began to be expressed at 5 weeks, and its expression increased markedly at 7 weeks after implantation. At 4-5 weeks after implantation, new bone adjacent to the membrane filters and cartilage toward the center of the diffusion chamber were observed histochemically. Light microscopic and immunohistologic examinations of chambers with marrow cells and DBM revealed production of mineralized matrices, typical of bone characterized by the appearance of BGP and mineralized nodules. In contrast, bone marrow cells alone did not show extensive bone formation and yielded very low values for these biochemical parameters. The present experiments demonstrate the potential of bone marrow cells and DBM to produce not only cartilage formation but also membranous bone formation associated with increasing expression of BGP mRNA during the later stages of bone formation, as well as a marked accumulation of BGP.

Osteogenic phenotype expression of allogeneic rat marrow cells in porous hydroxyapatite ceramics.

Porous hydroxyapatite (HA) ceramics were combined with either allogeneic (ACI) or isogeneic (Fischer 344) rat marrow cells and implanted in subcutaneous sites of Fischer rats. FK506 as an immunosuppressant or saline was administered to the recipient rats. The implanted marrow/HA composites were harvested on day 28 and analyzed for bone-forming capability by determining osteoblastic phenotype expression levels of protein synthesis and gene expression. The alkaline phosphatase (ALP) activity and osteocalcin (OC) contents were very low and mRNAs (Northern blot analysis) were not detected in the allografts without FK506. However, high activity of ALP and high content of OC were found and mRNAs were detected in the allografts with FK506 and in the isografts (with and without FK506). This analysis indicates the osteogenic potential of allogeneic marrow cells in the presence of FK506. The histologic sections revealed that allografts without FK506 did not show bone formation but did show the infiltration of many small cells in the ceramics indicating an immunologic reaction, however, the allografts with FK506 and the isografts (with and without FK506) showed consistent de novo bone formation on the HA pore surface. These results indicate that FK506 can suppress the immunologic reaction in the allografts and induce a favorable conditions to support osteoblastic differentiation of allogeneic rat marrow stromal stem cells on the surface of HA ceramics. Therefore, our study suggests the feasibility of clinical transplantation of allogeneic bone marrow for a selected bone graft in applications using adjuvant systemic immunosuppression.

A novel polymorphism in the promoter region for the human osteocalcin gene: the possibility of a correlation with bone mineral density in postmenopausal Japanese women.

We present a polymorphism of the human osteocalcin gene (also known as BGP, for bone Gla protein) due to a 1 base pair (bp) substitution from cytosine to thymine at position 298 nucleotides (nt), which is at position 198 nt upstream from the BGP exon 1. This mutation was detected by single-strand conformation polymorphism analysis after polymerase chain reaction for the osteocalcin gene fragment (326 bp) and sequencing analysis. The cytosine/thymine polymorphism can be defined by restriction fragment length polymorphism analysis using a modified primer pair and the restriction endonuclease HindIII. The osteocalcin genotype was determined in 160 postmenopausal Japanese women (age 48-80 years). Osteocalcin alleles were designated according to the absence (H) or presence (h) of the HindIII restriction site. There were 12 HH, 49 Hh, and 99 hh individuals, and the allele frequencies were 22.8% for H and 77.2% for h. To determine if genetic variation influences bone mineral density (BMD) and thus can be a determinant of susceptibility to osteoporosis in older women, we examined the association of BMD with the osteocalcin genotypes found in the present study. The subjects with genotype HH had the smallest BMD and those with hh had the greatest BMD among subjects, but these differences did not reach statistical significance. The HindIII genotype showed a significant effect on the prevalence of osteopenia in the subjects, that is, women with genotype HH had a 5.74 times greater risk for osteopenia (p < 0.05) and those with genotype Hh had a 1.59 times greater risk than women with genotype hh. We identified the osteocalcin gene polymorphism, detected with the HindIII genotype, which was suggested to influence bone density and is a possible genetic marker for bone metabolism.

In vitro chondrogenesis stimulated by extracts of gall bladder epithelium.

Living adult gall bladder epithelium has previously been shown to initiate in vivo ectopic cartilage and/or bone formation. In the present study, we demonstrate that solubilized extracts of gall bladder, which have never been inductive when tested in vivo, do in fact contain a factor that stimulates chondrogenesis in stage 24 chick limb bud mesenchymal cell cultures. These 4 M guanidinium chloride soluble, cold water soluble protein fractions initiate a dose-dependent increase in the amount of cartilage formed following an 8-10 day continuous exposure as evidenced by the presence of greater numbers of chondrocytes when viewed with phase optics, more intense toluidine blue metachromatic staining in fixed plates and elevated 35S-sulfate incorporation into the cell layer (as a maker for proteoglycan biosynthesis). The factor(s) responsible for this activity binds to DEAE anion-exchange resins, is stable to heat (100 degrees C for 10 min) and 2-mercaptoethanol reduction but is labile to trypsin digestion. These results emphasize the importance of in vitro test systems to identify bioactive agents which may otherwise go undetected using an in vivo implantation assay.

In vivo osteogenic durability of cultured bone in porous ceramics: a novel method for autogenous bone graft substitution.

BACKGROUND: Bone marrow cells differentiate into bone-forming osteoblasts when cultured in medium supplemented with 15% fetal bovine serum, ascorbic acid, beta-glycerophosphate, and dexamethasone. METHODS: To investigate in vivo osteoblastic activity and bone matrix formation by cultured bone marrow cells, Fischer rat marrow cells were cultured for 2 weeks in porous hydroxyapatite (HA) and then subcutaneously implanted into 7-week-old male syngeneic rats. The implants were harvested after 8 and 52 weeks for biochemical and histological analyses. RESULTS: At both times, formation of lamellar bone accompanied by regeneration of marrow were seen in many of the HA pores. When a fluorochrome (calcein) was administered at 50 weeks after implantation, it was detected in the pores of implants harvested at 52 weeks. Osteoclastic resorption followed by new bone formation was seen in some pores at 52 weeks, indicating that bone remodeling was continuing. The alkaline phosphatase activity of implants harvested at 52 weeks was comparable to that at 8 weeks, whereas the osteocalcin content of the implants harvested at 52 weeks was about twice that at 8 weeks. CONCLUSION: These results demonstrated that there was persistent in vivo osteogenic and hematopoietic activity in the prefabricated bone/HA constructs, and indicated that normal bone tissue was regenerated after grafting of the constructs, which were brittle before implantation. Tissue engineering using HA and cultured marrow cells culture may provide an alternative method of bone transplantation for patients with skeletal disorders, although further in vivo and in vitro experiments are needed.

Bonding osteogenesis in coralline hydroxyapatite combined with bone marrow cells.

Coralline hydroxyapatite ceramics alone (control) and the ceramics combined with rat marrow cells were implanted subcutaneously in the backs of syngeneic Fischer rats and harvested at 1,2,3,4,6,8 and 24 wk after surgery. None of the control ceramics (without marrow) showed bone formation. However, ceramics combined with marrow cells showed consistent new bone formation in the pore regions. Histometrical results revealed increased new bone formation over time. Undecalcified sections of the ceramics studied by fluorochrome labelling showed that the osteogenesis began directly on the surface of the ceramic and proceeded centripetally towards the centre of the pores (bonding osteogenesis). SEM-EPMA analysis of the bone-ceramic interface also revealed direct bonding of bone to the ceramic surface.

Histological and biochemical evaluation of osteogenic response in porous hydroxyapatite coated alumina ceramics.

We compared the osteogenic response in porous alumni (Al) and hydroxyapatite coated porous alumni ceramic (HA/Al) by grafting rat bone marrow cells with these implants subcutaneously in the back of syngeneic rats. The ceramics did not show any bone formation without marrow, but did when combined with marrow cells. The osteogenesis in HA/Al began directly on the ceramic surface, but in Al began away from the ceramic surface, and fibrous tissue interposition was seen between the de novo bone and its surface. Alkaline phosphatase activity and bone specific bone gla protein content in HA/Al with marrow were both about three times higher than in Al with marrow. These results indicate that HA/Al has excellent osteoconductive properties and that a composite of marrow and HA/Al is clinically applicable osteogenic biomaterial.

Osteogenesis coordinated in C3H10T1/2 cells by adipogenesis-dependent BMP-2 expression system.

A novel tissue engineering for bone formation has been proposed, to make osteoblast differentiation balanced by transfecting the mesenchymal stem cells with a gene encoding human bone morphogenetic protein-2 (hBMP-2) under the control of adipocyte specific lipoprotein lipase (LPL) promoter. Due to the promoter specificity, the initiation of BMP transcription is dependent on adipogenesis. For 14-day culture in the presence of ascorbic acid (asc) and beta-glycerophosphate (gly), nontransfected mouse embryonic fibroblast C3H10T1/2 (10T1/2) cells showed extensive accumulation of lipid droplets and adipocyte specific enzyme glycerol-3-phosphate dehydrogenase (G3PDH) mRNA expression, but exhibited neither BMP-2 expression, high alkaline phosphatase (ALP) activity which reflects osteoblast phenotype. On the other hand, transfected 10T1/2 cells showed hBMP-2 expression, high ALP activity and low level of G3PDH. mRNA expression accompanied with minimal lipid droplets. These results indicate that 10T1/2 cells are proved to be differentiated with maintaining coordinated balance of adipogenesis and osteogenesis, when they are transfected by the gene encoding hBMP-2 under the control of LPL promoter.

Increased expression of extracellular signal regulated kinase 1 after axotomy in the dorsal motor nucleus of the vagus nerve and the hypoglossal nucleus.

The extracellular signal regulated kinases (Erks) cascade is a major signalling system by which cells transduce extracellular signals into intracellular responses. To obtain information about the role of Erks in retrograde neuronal reaction, we investigated the changes of Erk 1 and Erk 2 with in situ hybridization and immunohistochemical study in the dorsal motor nucleus of vagus nerve, which shows degenerative changes, and the hypoglossal nucleus, which shows regenerative changes, of adult rats after axotomy. The expression of mRNA and protein of Erk 1 increased between 7 and 28 days after axotomy both in the vagal and hypoglossal nuclei, however, there was no remarkable change in those of Erk 2. The increased expression of Erk 1 is common to both regenerative hypoglossal and degenerative vagal neurons. These findings indicate that Erk 1 is closely related with the retrograde neuronal reaction but whether neurons are destined to survive or die depends on some other factors.

Viability of ischemia/reperfused muscles in rat: a new evaluation method by RNA degradation.

The rat's skeletal muscle viability was evaluated using the muscle viability index (MVI) which reflects the mRNA degradation. To evaluate ischemic injury of the muscle, 24 hind limbs of Fischer rats (three subgroups of eight rats each) were preserved at normothermia for 1, 3 and 6 h and then tibialis anterior muscle was harvested. To investigate ischemia/reperfusion injury, another 48 limbs were transplanted to recipient Fischer rats after the ischemia at normothermia for 1, 3 and 6 h, respectively. The transplanted muscles were harvested on day 3 and day 7 after transplantation. Eight fresh muscles were also harvested and used as control. Total RNA isolated from each muscle was fractionated by electrophoresis and hybridized with 32P-labelled cDNA of GAPDH, and the radioactivity of intact and degraded GAPDH mRNA was measured. MVI was calculated as follows, MVI = [X/(X + Y)] x 100, where X and Y represent the radioactivities corresponding to intact GAPDH and degraded GAPDH mRNA band, respectively. In 1-h ischemia group, the MVI indices of both ischemic insult and ischemia/reperfusion group were comparable to control. In the 3-h ischemia group, the index of ischemia/reperfused group was comparable to control although the index of ischemic insult group was significantly lower than control. However, in the 6-h ischemia group, both indices of ischemic insult and ischemia/reperfusion group were significantly lower than control. These results show that the muscle damage was detected in ischemia at normothermia even after 3 h. However, this damage was overcome by reperfusion. There was no recovery from damage in muscles that had been preserved for more than 6 h which had resulted in irreversible degeneration. Therefore, in clinical muscle transplantation, one has to transplant the muscle at least within 3-h ischemia.

Osteogenic potential of allogeneic rat marrow cells in porous hydroxyapatite ceramics: a histological study.

Hydroxyapatite ceramics facilitate osteogenesis but cannot induce bone formation by themselves. We studied the feasibility of bone formation supported by allogeneic bone marrow cells in porous hydroxyapatite ceramics. Coralline hydroxyapatite discs were soaked in a marrow cell suspension harvested from either ACI (RT1a), Lewis (RT1(1)), or Fischer 344 (RT1(1v)) male rats, and these discs were implanted subcutaneously into 56 male Fischer 344 rats. FK-506 (tacrolimus hydrate), an immunosuppressant, or saline was injected intramuscularly into the recipients every day for 2 weeks after surgery, and additional injections were given to 19 of the rats every 2 days for 2 more weeks. Neither of the mismatched major (ACI rat) or minor (Lewis rat) marrow cell transplants showed any bone formation without administration of FK-506. However, in rats treated with FK-506, bone formed in the pores of all the three types of ceramics implanted, which each contained the marrow cells from one of the three kinds of rats used. There were no differences among the three groups of donors with regard to the bone formation ratio. We previously reported that subcutaneous implantation of porous hydroxyapatite combined with isogeneic marrow cells resulted in consistent bone formation, even at ectopic sites. Since it would be difficult to harvest a large number of autologous marrow cells in clinical cases, we attempted to use allogeneic marrow cells and have shown the allogeneic murine marrow cells to have osteogenic potential.

Heterotopic osteogenesis in porous ceramics induced by marrow cells.

When untreated porous calcium phosphate ceramics were transplanted into subcutaneous (s.c.) or intramuscular (i.m.) sites, fibrovascular tissue grew in the pore region without evidence of bone formation. However, when these same ceramics were combined with syngeneic marrow cells, osteogenesis was observed inside the pore region of the implanted ceramic. The osteogenesis began on the surface of the pore region at approximately 3 weeks postimplantation by a process of intramembranous bone formation, with the de novo bone tissue observed directly interfacing with the ceramic surface. Infrequently, small isolated areas showed cartilage formation with no noticeable endochondral ossification. At 4 weeks postimplantation of the ceramic with marrow cells, the osteogenesis in the ceramic accompanied an observed increase in compressive strength, rigidity, and energy absorption of the ceramic. These results suggest that a combination of porous ceramics and marrow cells may be useful for clinical problems requiring osseous reconstruction.

Histologic and biochemical analysis of osteogenic capacity of vascularized periosteum.

We analyzed the osteogenic capacity of the vascularized periosteum histologically and biochemically using a new experimental model of the vascularized tibial periosteum-hydroxyapatite composite in rats. Bone formation was observed not only on the surface but also in the pores of the hydroxyapatite at 4 weeks. The alkaline phosphatase activity increased to a peak at 2 weeks, with half this activity maintained until 8 weeks. The bone-specific bone gla protein content increased constantly as time passed. On the other hand, in the vascularized fascia-hydroxyapatite composite group (control group), no bone formation was observed histologically, alkaline phospatase activity was low, and bone gla protein content was very low. These results indicate that (1) the vascularized periosteum has the most significant osteogenic capaacity at 2 weeks, with a constant level of the activity maintained thereafter, (2) it forms new bone soon after operation, and (3) the amount of bone increases as time passes.

Relationship between meniscal degeneration and element contents.

The purpose of this study is to investigate the relationship between meniscal degeneration and element contents. The contents of elements (calcium, phosphorus, sulfur, and magnesium) in the menisci from 17 patients with osteoarthritis (OA) of the knee, 6 with rheumatoid arthritis (RA), and 2 who underwent the surgical operation for malignant tumors (control) were analyzed by inductively coupled plasma-atomic emission spectrometry, and the menisci were divided into four stages (Stage 0-3) of histological degeneration. The calcium contents of the menisci were 0.26 +/- 0.16 in Stage 0, 0.50 +/- 0.37 in Stage 1, and 0.69 +/- 0.66 in Stage 2, respectively (the values represent mg elements/g dry tissue). They increased with the progression of the stage. This tendency was found in the menisci with OA, but was not clear in those with RA. The calcium content in the control group was 0.17 +/- 0.09 mg/g. There was no significant relationship between the stage of degeneration and the contents of phosphorus, sulfur, or magnesium. The calcium content of the meniscus might indicate the degree of meniscal degeneration.

Osteogenic differentiation of cultured marrow stromal stem cells on surface of microporous hydroxyapatite based mica composite and macroporous synthetic hydroxyapatite.

In order to investigate the significance of hydroxyapatite based microporous composite (HA/mica composite) surfaces and a macroporous synthetic hydroxyapatite, rat marrow cell culture, which shows osteogenic differentiation, was carried out on six different culture substrata (two control culture dishes, two identical HA/mica composites, and two identical macroporous synthetic hydroxyapatites). A culture period of two weeks in the presence of beta-glycerophosphate (BGP), ascorbic acid, and dexamethasone resulted in abundant mineralized nodule formations that were positive for alkaline phosphatase (ALP) stain. The stain on the macroporous synthetic hydroxyapatite and the HA/mica composites were intense, the enzyme activity being about double that of control culture dishes. These data indicate that the synthetic macroporous hydroxyapatite surface and the HA/mica composite surface promotes osteoblastic differentiation.