Short-term follow-up of disc cell therapy in a porcine nucleotomy model with an albumin-hyaluronan hydrogel: in vivo and in vitro results of metabolic disc cell activity and implant distribution.

PURPOSE: Cell therapy would be favorably performed immediately after nucleotomy, to restore intervertebral disc functionality and to slow down disc degeneration. Promising results were reported from small animal models but remaining problems, especially in larger animals, include loss of vital cells due to annular damage at the injection site and detrimental intradiscal conditions. The aim of the present study was to optimize cell-based disc therapy using a new albumin-hyaluronan hydrogel together with bone marrow-derived mesenchymal stem cells in a large porcine disc model. METHODS: Luciferase cell labeling was evaluated to follow-up stem cells metabolically up to 7 days in 3D cell cultures mimicking the harsh disc environment with low oxygen and glucose concentrations. As a pilot in vivo study, the implant was injected into porcine discs after removal of ~10% of nucleus volume and animals were killed immediately after surgery (n = 6) and 3 days later (n = 6). 24 discs were analyzed. Implant persistence and cell activity (luciferase + WST assay) were observed simultaneously. RESULTS: In vitro cell culture with reduction of glucose (20, 5, 0.5, 0 mM) and oxygen (21, 5, 2%) significantly decreased metabolic cell activity and luciferase activity after 3 days, with no recovery and a further decrease after 7 days, establishing luciferase activity as a metabolic sensor. During 3 days of 3D culture with disc-like conditions, luciferase activity decreased to 8%. In vivo, initial implant volume shrank to 61% at day 3 with evidence for hydrogel compression. Luciferase activity in vivo at day 3 was 2% without referencing but 23% after referencing to in vitro cell adaptation, and 38% after additional consideration of detected implant volume loss. CONCLUSION: In vitro analysis up to 7 days established for the first time luciferase activity as a metabolic sensor for mesenchymal stem cells used in regenerative disc therapy. Under the present protocol, short-term in vivo analysis after 3 days suggests improved implant retainment inside the disc and persistence of metabolically active cells; however, further studies will have to prove long-term in vivo outcome.

In vivo and in vitro investigations of heterozygous nebulin knock-out mice disclose a mild skeletal muscle phenotype.

Nemaline myopathy is the most common congenital skeletal muscle disease, and mutations in the nebulin gene account for 50% of all cases. Recent studies suggest that the disease severity might be related to the nebulin expression levels. Considering that mutations in the nebulin gene are typically recessive, one would expect that a single functional nebulin allele would maintain nebulin protein expression which would result in preserved skeletal muscle function. We investigated skeletal muscle function of heterozygous nebulin knock-out (i.e., nebulin(+/-)) mice using a multidisciplinary approach including protein and gene expression analysis and combined in vivo and in vitro force measurements. Skeletal muscle anatomy and energy metabolism were studied strictly non-invasively using magnetic resonance imaging and 31P-magnetic resonance spectroscopy. Maximal force production was reduced by around 16% in isolated muscle of nebulin(+/-) mice while in vivo force generating capacity was preserved. Muscle weakness was associated with a shift toward a slower proteomic phenotype, but was not related to nebulin protein deficiency or to an impaired energy metabolism. Further studies would be warranted in order to determine the mechanisms leading to a mild skeletal muscle phenotype resulting from the expression of a single nebulin allele.

Methods to monitor distribution and metabolic activity of mesenchymal stem cells following in vivo injection into nucleotomized porcine intervertebral discs.

Intervertebral disc (IVD) degeneration involves a series of biochemical and morphological changes leading to loss of spinal stability and flexibility. Cell therapy is promising to reconstitute IVDs with new cells, however, sustained metabolic activity seems crucial for an active contribution to regeneration. The aim of the present study was to establish methods for separate follow up of persistence and activity of autologous porcine mesenchymal stem cells (pMSC) after implantation into IVDs of Goettingen minipigs in vivo in order to conclude about the potential of such an intervention strategy. For quantitative follow up, the transfer matrix was supplemented with Al(2)O(3) particles and pMSC which were retrovirally labeled with firefly luciferase (pMSC-Luc). Six mature Goettingen minipigs underwent matrix based cell transfer after partial nucleotomy of lumbar IVDs (n = 24). Day 0 and day 3 segments were analyzed for retained volume of Al(2)O(3) particles by micro-computed-tomography (muCT) and for cell activity by luciferase enzyme assessment. Three days after injection a reduction of Al(2)O(3) particles (P = 0.028) to about 9% and of pMSC-Luc activity to about 7% of initial values (P = 0.003) was detected, which suggests loss of 90% of the implant material under in vivo conditions without evidence for reduced pMSC-Luc metabolic activity (P = 0.887). In conclusion, separate follow up of implant material and cell activity was possible and unravels problems with in vivo implant persistence after annular puncture rather than quick loss of cell activity. Therefore, IVD-regeneration-strategies should increasingly focus on annulus reconstruction in order to reduce implant loss due to annular failure.