Quantification of chromatographic effects of vitamin B supplementation in urine and implications for hydration assessment.

Changes in body water elicit reflex adjustments at the kidney, thus maintaining fluid volume homeostasis. These renal adjustments change the concentration and color of urine, variables that can, in turn, be used as biomarkers of hydration status. It has been suggested that vitamin supplementation alters urine color; it is unclear whether any such alteration would confound hydration assessment via colorimetric evaluation. We tested the hypothesis that overnight vitamin B2 and/or B12 supplementation alters urine color as a marker of hydration status. Thirty healthy volunteers were monitored during a 3-day euhydrated baseline, confirmed via first morning nude body mass, urine specific gravity, and urine osmolality. Volunteers then randomly received B2 (n = 10), B12 (n = 10), or B2 + B12 (n = 10) at ∼200 × recommended dietary allowance. Euhydration was verified on trial days (two of the following: body mass ± 1.0% of the mean of visits 1-3, urine specific gravity < 1.02, urine osmolality < 700 mmol/kg). Vitamin purity and urinary B2 concentration ([B2]) and [B12] were quantified via ultraperformance liquid chromatography. Two independent observers assessed urine color using an eight-point standardized color chart. Following supplementation, urinary [B2] was elevated; however, urine color was not different between nonsupplemented and supplemented trials. For example, in the B2 trial, urinary [B2] increased from 8.6 × 10(4) ± 7.7 × 10(4) to 5.7 × 10(6) ± 5.3 × 10(6) nmol/l (P < 0.05), and urine color went from 4 ± 1 to 5 ± 1 (P > 0.05). Both conditions met the euhydrated color classification. We conclude that a large overnight dose of vitamins B2 and B12 does not confound assessment of euhydrated status via urine color.

Meniscal repair using engineered tissue.

In this study, devitalized meniscal tissue pre-seeded with viable cultured chondrocytes was used to repair a bucket-handle incision in meniscal tissue transplanted to nude mice. Lamb knee menisci were devitalized by cyclic freezing and thawing. Chips measuring four by two by one-half millimeters were cut from this devitalized tissue to serve as scaffolds. These chips were then cultured either with or without viable allogeneic lamb chondrocytes. From the inner third of the devitalized meniscal tissue, rectangles were also cut approximately 8 x 6 mm. A 4 mm bucket-handle type incision was made in these blocks. The previously prepared chips either with (experimental group) or without viable chondrocytes (control group) were positioned into the incisions and secured with suture. Further control groups included blocks of devitalized menisci with incisions into which no chips were positioned and either closed with suture or left open with no suture. Specimens were transplanted to subcutaneous pouches of nude mice for 14 weeks. After 14 weeks, seven of eight experimental specimens (chips with viable chondrocytes) demonstrated bridging of the incision assessed by gross inspection and manual distraction. All the control groups were markedly different from the experimental group in that the incision remained grossly visible. Histological analysis was consistent with the differences apparent at the gross level. Only the experimental specimens (chips with viable chondrocytes) with gross bridging demonstrated obliteration of the interface between incision and scaffold. None of the control specimens revealed any cells or tissue filling the incision. Tissue engineering using scaffolds and viable cells may have an application in meniscal repair in vivo.

Mitotic activity in chondrocyte transplantation from the in vitro phase to the in vivo phase.

The transplantation of devitalized allogenic matrices vehiculating autologous chondrocytes, previously isoled and seeded on them could be a solution to the problem of repairing lesions of the joint cartilage. For the matrix/cell "composite" to be "graftable" the cells must continue to duplicate and produce cartilaginous matrix even after transport in vivo. The present study analyzes the mitotic activity of chondrocytes planted on devitalized allogenic cartilage and grafted in living animals. Chondrocytes of joint cartilage of lambs were isolated enzymatically and then seeded in vitro on devitalized allogenic cartilaginous matrices for 3 weeks. At the end of the co-culture period, these matrix/chondrocyte composites were transplanted in subcutaneous pockets of athymic mice. The experimental and control samples were evaluated subsequent to explantation by histological study and incorporation of tritiated thymidine. The results obtained revealed an important decrease in the values for the incorporation of thymidine beginning from experimental time 0 (pre-implant evaluation) up to day 28 after implantation, followed by a mild increase at the experimental time of 42 days. This study demonstrated the tendency of articular chondrocytes cultivated in vitro and subsequently transplanted in vivo on a support of devitalized allogenic cartilaginous matrix to modify mitotic activity from very high values for the first experimental times, typical of the in vitro phases of cellular expansion, to very low values, more similar to the behavior of articular chondrocytes in vivo.

Biomechanical analysis of a chondrocyte-based repair model of articular cartilage.

The objective of this study was to evaluate the biomechanical properties of newly formed cartilaginous tissue synthesized from isolated chondrocytes. Cartilage from articular joints of lambs was either digested in collagenase to isolated chondrocytes or cut into discs that were devitalized by multiple freeze-thaw cycles. Isolated cells were incubated in suspension culture in the presence of devitalized cartilage matrix for 3 weeks. Multiple chondrocyte/matrix constructs were assembled with fibrin glue and implanted subcutaneously in nude mice for up to 6 weeks. Testing methods were devised to quantify integration of cartilage pieces and mechanical properties of constructs. These studies showed monotonic increase with time in tensile strength, fracture strain, fracture energy, and tensile modulus to values 5-10% of normal articular cartilage by 6 weeks in vivo. Histological analysis indicated that chondrocytes grown on dead cartilage matrix produced new matrix that integrated individual cartilage pieces with mechanically functional tissue.

Recapitulation of signals regulating embryonic bone formation during postnatal growth and in fracture repair.

A number of proteins have recently been identified which play roles in regulating bone development. One important example is Indian hedgehog (Ihh) which is secreted by the prehyprtrophic chondrocytes. Ihh acts as an activator of a second secreted factor, parathyroid hormone-related protein (PTHrP), which, in turn, negatively regulates the rate of chondrocyte differentiation. Here we examine the expression of these genes and their molecular targets during different stages of bone development. In addition to regulating PTHrP expression in the perichondrium, we find evidence that Ihh may also act on the chondrocytes themselves at particular stages. As bone growth continues postnatally in mammals and the developmental process is reactivated during fracture repair, understanding the molecular basis regulating bone development is of medical relevance. We find that the same molecules that regulate embryonic endochondral ossification are also expressed during postnatal bone growth and fracture healing, suggesting that these processes are controlled by similar mechanisms.

Bonding of cartilage matrices with cultured chondrocytes: an experimental model.

The capacity of isolated chondrocytes to join separate masses of cartilage matrix was investigated with composites implanted in subcutaneous pouches in nude mice. Slices of articular cartilage were harvested from lambs and were devitalized by cyclic freezing and thawing. The slices were then either co-cultured with viable allogeneic lamb chondrocytes (experimental) or cultured without such chondrocytes (control). Composites of three slices were constructed with use of fibrin glue and were implanted in nude mice for periods ranging from 7 to 42 days. Bonding of the experimental matrices with viable chondrocytes was achieved at 28 and 42 days, as assessed by direct examination, histology, thymidine uptake, and fluorescence. No bonding occurred in the control composites without viable chondrocytes. We conclude that devitalized cartilage matrix is a scaffold to which isolated chondrocytes can attach and begin to repopulate.

Repopulation of laser-perforated chondroepiphyseal matrix with xenogeneic chondrocytes. An experimental model.

Growth of chondrocytes into a xenogeneic chondroepiphyseal matrix was investigated in an in vitro experimental model by combining viable calf chondrocytes with chick epiphyseal matrix devoid of viable chondrocytes. The chondrocytes were harvested from the wrist joints of newborn calves and cultured for 2 days. The epiphyses were harvested from the distal femurs and the proximal tibias of fetal chicks after development was arrested at 17 days by freezing. The epiphyseal specimens were prepared in four ways. These included femoral and tibial epiphyses without holes and femoral and tibial epiphyses with holes made by a laser. These epiphyseal specimens were co-cultured with calf chondrocytes for various periods. After digestion of the epiphyseal matrix, viable chondrocytes were counted in suspension. Chondrocyte division in the matrix was assessed by [3H]thymidine incorporation. The growth of calf chondrocytes into the xenogeneic chick matrix was evaluated by fluorescence microscopy on fresh thick epiphyseal sections. The percentage of viable chondrocytes in the xenogeneic epiphyseal matrix increased with culture time to a maximum at day 21. The addition of laser-drilled holes was found to extend a plateau of chondrocyte viability until day 29. A decrease in cell viability was detected at later observation points. This study demonstrates that xenogeneic matrix may serve as a morphogenetic scaffold for chondrocytic growth.

Laboratory tests in rheumatology.

The most commonly used laboratory tests in the diagnosis and monitoring of rheumatic diseases are reviewed. Particular emphasis is placed on antinuclear antibodies as markers of specific rheumatic disorders and disease subsets. The use of synovial fluid tests to differentiate between inflammatory and noninflammatory rheumatic disorders is described as well as the use of common hematological tests.