Genetic engineering of juvenile human chondrocytes improves scaffold-free mosaic neocartilage grafts.

BACKGROUND: Current cartilage transplantation techniques achieve suboptimal restoration and rely on patient donor cells or living grafts of chondrocytes. PURPOSE: We sought to enhance allogeneic grafts by testing mosaics of genetically engineered and naïve juvenile human chondrocytes (jCh). METHODS: We obtained specimens from three humans and performed three experiments (two in vitro, one in vivo). We compared neocartilage with and without (1) supplemented serum-free medium (chondrocyte differentiation medium [CDM]), (2) adenoviral BMP-2 (AdBMP-2) transduction, and (3) varying ratios (0.1-1) of transduced and naïve jCh. We compared (4) healing with mosaic grafts with naïve neocartilage or marrow stimulation in immunosuppressed rats. For each of 10 in vitro treatment groups, we had six replicates for each human, and for each of three in vivo treatment groups, we had four replicates for one human. We scored the histology with the semiquantitative Bern score. RESULTS: AdBMP-2 and naïve neocartilage growth in CDM were histologically superior (Bern score, 5.2 versus 3.7; 8.0 versus 1.8) and size (8.0 versus 6.1; 7.9 versus 2.2 mg) to standard medium. In CDM, AdBMP-2 decreased viability (76% versus 90%), but increased BMP-2 production (619 ng/mL versus 43 pg/mL). Ten percent and 25% AdBMP-2 transduction had Bern scores of 6.8 and 6.5 and viability of 84% and 83%, respectively. Twenty-five percent mosaic grafts provided better healing histologically than marrow stimulation or naive neocartilage. CONCLUSIONS: Low-level AdBMP-2 and CDM augment neocartilage parameters in vitro and vivo. CLINICAL RELEVANCE: Genetic augmentation of jCh and creation of mosaic neocartilage may improve graft viability and articular healing compared with naïve neocartilage.

Fibroblast growth factor 2-stimulated proliferation is lower in muscle precursor cells from old rats.

In aged skeletal muscle, impairments in regrowth and regeneration may be explained by a decreased responsiveness of muscle precursor cells (MPCs) to environmental cues such as growth factors. We hypothesized that impaired responsiveness to fibroblast growth factor 2 (FGF2) in MPCs from old animals would be explained by impaired FGF2 signalling. We determined that 5-bromo-2'-deoxyuridine (BrdU) incorporation and cell number increase less in MPCs from 32- compared with 3-month-old rats. In the presence of FGF2, we demonstrated that there were age-associated differential expression patterns for FGF receptor 1 and 2 mRNAs. Measurement of downstream signalling revealed that that mitogen-activated protein kinase/ERK kinase 1/2 (MEK1/2)-extracellular signal-regulated kinase 1/2, protein kinase C and p38 were FGF2-driven pathways in MPCs. Uniquely, protein kinase C signalling was shown to play the largest role in FGF2-stimulated proliferation in MPCs. c-Jun N-terminal kinase (JNK) signalling was ruled out as an FGF2-stimulated proliferation pathway in MPCs. Inhibition of JNK had no effect on FGF2 signalling to BrdU incorporation, and FGF2 treatment was associated with increased phosphorylation of p38, which inhibits, rather than stimulates, BrdU incorporation in MPCs. Surprisingly, the commonly used vehicle, dimethyl sulphoxide, rescued proliferation in MPCs from old animals. These findings provide insight for the development of effective treatment strategies that target the age-related impairments of MPC proliferation in old skeletal muscle.

Postmortem alterations in the pH range of myofibrillar ATPase activation/inactivation.

A histochemical assay for myofibrillar adenosine triphosphatase (mATPase) activity is routinely utilized in the delineation of fiber types in healthy human skeletal muscle. Each fiber type has a specific pH range of mATPase stability (activation). Outside of this pH range, mATPase activity is labile (inactivated), no reaction product is formed, and the fibers remain unstained. The aim of the present study was to carefully investigate the pH stability/lability of mATPase in postmortem muscles. To this end, vastus lateralis muscle samples were obtained approximately 0.5, 1, 2, 3, and 4 days after death, as well as control samples from a healthy young man and woman. Serial cross sections of the muscle samples were assayed for mATPase activity throughout preincubation pH ranges of 4.15-4.7 and 10.2-10.5 in increments of 0.05 pH units. Myosin heavy chain analysis (as well as a regression analysis comparing fiber type area and relative myosin heavy chain content) verified the mATPase-based fiber types. The pH ranges of mATPase stability/lability for the control samples were as previously reported, and support the use of preincubation pH values of 4.3, 4.6, and 10.4 for the delineation of fiber types in normal human muscle. For the postmortem samples, both quantitative and qualitative changes altered the pH ranges of mATPase activation/inactivation. Quantitative changes consisted of a time-dependent loss of mATPase activity that was inhibited in all fibers outside the pH range of 4.15-10.50. In addition, qualitative changes caused "shifts to the left" in mATPase stability within the fast fiber types (IIA and IIB). As such, complete inhibition of mATPase activity did not occur until preincubation at pH 4.45 and pH 4.30 for fiber types IIA and IIB, respectively. For the postmortem vastus lateralis muscle samples, optimal preincubation pH values for mATPase-based fiber type delineation were pH 4.30, 4.45, and 10.35. The reason for these qualitative changes in mATPase stability is not known. However, postmortem changes such as increased lactate production and marked acidification may play a role.