Enzyme replacement therapy for murine hypophosphatasia.

INTRODUCTION: Hypophosphatasia (HPP) is the inborn error of metabolism that features rickets or osteomalacia caused by loss-of-function mutation(s) within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNALP). Consequently, natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5'-phosphate (PLP), a co-factor form of vitamin B6. Babies with the infantile form of HPP often die with severe rickets and sometimes hypercalcemia and vitamin B6-dependent seizures. There is no established medical treatment. MATERIALS AND METHODS: Human TNALP was bioengineered with the C terminus extended by the Fc region of human IgG for one-step purification and a deca-aspartate sequence (D10) for targeting to mineralizing tissue (sALP-FcD10). TNALP-null mice (Akp2-/-), an excellent model for infantile HPP, were treated from birth using sALP-FcD10. Short-term and long-term efficacy studies consisted of once daily subcutaneous injections of 1, 2, or 8.2 mg/kg sALP-FcD10 for 15, 19, and 15 or 52 days, respectively. We assessed survival and growth rates, circulating levels of sALP-FcD10 activity, calcium, PPi, and pyridoxal, as well as skeletal and dental manifestations using radiography, microCT, and histomorphometry. RESULTS: Akp2-/- mice receiving high-dose sALP-FcD10 grew normally and appeared well without skeletal or dental disease or epilepsy. Plasma calcium, PPi, and pyridoxal concentrations remained in their normal ranges. We found no evidence of significant skeletal or dental disease. CONCLUSIONS: Enzyme replacement using a bone-targeted, recombinant form of human TNALP prevents infantile HPP in Akp2-/- mice.

The effects of RANKL inhibition on fracture healing and bone strength in a mouse model of osteogenesis imperfecta.

Currently, the standard treatment for osteogenesis imperfecta (OI) is bisphosphonate therapy. Recent studies, however, have shown delayed healing of osteotomies in a subset of OI patients treated with such agents. The current study sought to determine the effects of another therapy, RANKL inhibition, on bone healing and bone strength in the growing oim/oim mouse, a model of moderate to severe OI. Mice [73 oim/oim and 69 wild-type (WT)] were injected twice weekly with either soluble murine RANK (RANK-Fc) (1.5 mg/kg) or saline beginning at 6 weeks of age. At 8 weeks of age, the animals underwent transverse mid-diaphyseal osteotomies of the right femur. Therapy was continued until sacrifice at 2, 3, 4, or 6 weeks postfracture. At 6 weeks post-fracture, greater callus area (6.59 +/- 3.78 mm(2) vs. 2.67 +/- 2.05 mm(2), p = 0.003) and increased radiographic intensity (mineral density) (0.48 +/- 0.14 vs. 0.30 +/- 0.80, p = 0.005) were found in the RANK-Fc versus saline oim/oim group, indicating a delay in callus remodeling. Despite this delay, mechanical tests at 6 weeks postfracture revealed no significant differences in whole bone properties of stiffness and failure moment. Further, RANKL inhibition resulted in a greater failure moment and greater work to failure for the nonfractured contralateral WT bones compared to the nonfractured saline WT bones. Together, these results demonstrate that RANKL inhibition does not adversely affect the mechanical properties of healing bone in the oim/oim mice, and is associated with increased strength in intact bone in the WT mice.

Increased resorptive activity and accompanying morphological alterations in osteoclasts derived from the oim/oim mouse model of osteogenesis imperfecta.

The current study addresses whether alterations in osteoclasts (OCs) derived from oim/oim mice, an established model of moderate-to-severe OI, are present. Bone marrow cells from oim/oim and wildtype (+/+) mice were cultured on bone slices in the presence of MCSF and RANKL and evaluated at days 0, 1, 2, 4, and 7. OCs were identified by tartrate-resistant acid phosphatase (TRAP) staining, and bone slice resorption pits were analyzed by reflection microscopy. Flow cytometry was used to examine CD51 (integrin alphaV) and CD61 (integrin beta3) markers. Confocal microscopy was used to assess changes in OC morphology and resorption. There was no difference between the OC precursors of the two genotypes in expression of CD51 and CD61 markers. At day 2, the bone slices seeded with oim/oim cells had a greater percentage of mononuclear cells associated with resorption pits compared to +/+ bone slices. At day 4, the diameter and area of oim/oim OCs were larger compared to the +/+ OCs, and the number of nuclei per OC was also greater for the oim/oim group. At day 7, the oim/oim OCs contained more F-actin rings compared to the +/+ OCs, and the number of OCs in the oim/oim group was greater compared to the +/+ group. The resorbed area of bone slices for the oim/oim group was also greater compared to the +/+ group at day 7. In conclusion, oim/oim mononuclear resorbing cells and OCs showed cellular changes and greater resorptive activity compared to +/+ cells, features that likely contribute to dysregulated bone remodeling in OI.

Optimal methods for the preservation of cartilage samples in MRI and correlative biochemical studies.

MRI studies of cartilage require the prevention of sample degradation before and during scanning and during shipment for correlative studies. Methods to achieve this include immersion in protease inhibitors (PIs), refrigeration, and freezing. In this study, bovine nasal cartilage (BNC) samples were stored in Dulbecco's phosphate-buffered saline (DPBS), DPBS with standard PIs, or PI solution with GM6001, a potent metalloproteinase inhibitor. For each buffer, three samples were scanned at +4 degrees C and stored at +4 degrees C or at -20 degrees C with thawing prior to imaging. T2 and magnetization transfer (MT) rate, km, were measured weekly over 4 months, after which time water and glycosaminoglycan (GAG) contents were compared with those of matching tissue excised pre-storage. Samples in DPBS exhibited increased T2 (+33.6% after 1 month at +4 degrees C, P = 0.040) and decreased km (-20.6%, P = 0.004), while refrigeration in DPBS with PI and GM6001 yielded good stability (T2: +2.7%, P = 0.874; km: -4.2%, P = 0.654 after 108 days at +4 degrees C). Water content increased while GAG content markedly decreased in all samples. Thus, stability in cartilage MRI parameters can be optimized with appropriate storage conditions, but storage time should nonetheless be minimized.

Fourier transform infrared imaging and MR microscopy studies detect compositional and structural changes in cartilage in a rabbit model of osteoarthritis.

Assessment of subtle changes in proteoglycan (PG) and collagen, the primary macromolecular components of cartilage, which is critical for diagnosis of the early stages of osteoarthritis (OA), has so far remained a challenge. In this study we induced osteoarthritic cartilage changes in a rabbit model by ligament transection and medial meniscectomy and monitored disease progression by infrared fiber optic probe (IFOP) spectroscopy, Fourier transform infrared imaging spectroscopy (FT-IRIS), and magnetic resonance imaging (MRI) microscopy. IFOP studies combined with chemometric partial least-squares analysis enabled us to monitor progressive cartilage surface changes from two to twelve weeks post-surgery. FT-IRIS studies of histological sections of femoral condyle cartilage revealed that compared with control cartilage the OA cartilage had significantly reduced PG content 2 and 4 weeks post-surgery, collagen fibril orientation changes 2 and 4 weeks post-surgery, and changes in collagen integrity 2 and 10 weeks post-surgery, but no significant changes in collagen content at any time. MR microscopy studies revealed reduced fixed charge density (FCD), indicative of reduced PG content, in the OA cartilage, compared with controls, 4 weeks post-surgery. A non-significant trend toward higher apparent MT exchange rate, k(m), was also found in the OA cartilage at this time point, suggesting changes in collagen structural features. These two MR findings for FCD and k(m) parallel the FT-IRIS findings of reduced PG content and altered collagen integrity, respectively. MR microscopy studies of the cartilage at the 12-week time point also found a trend toward longer T (2) values and reduced anisotropy in the deep zone of the OA cartilage, consistent with increased hydration and less ordered collagen. These studies reveal that FT-IRIS and MR microscopy provide complementary data on compositional changes in articular cartilage in the early stages of osteoarthritic degradation.

Fourier transform infrared imaging spectroscopy investigations in the pathogenesis and repair of cartilage.

Significant complications in the management of osteoarthritis (OA) are the inability to identify early cartilage changes during the development of the disease, and the lack of techniques to evaluate the tissue response to therapeutic and tissue engineering interventions. In recent studies several spectroscopic parameters have been elucidated by Fourier transform infrared imaging spectroscopy (FT-IRIS) that enable evaluation of molecular and compositional changes in human cartilage with progressively severe OA, and in repair cartilage from animal models. FT-IRIS permits evaluation of early-stage matrix changes in the primary components of cartilage, collagen and proteoglycan on histological sections at a spatial resolution of approximately 6.25 microm. In osteoarthritic cartilage, the collagen integrity, monitored by the ratio of peak areas at 1338 cm(-1)/Amide II, was found to correspond to the histological Mankin grade, the gold standard scale utilized to evaluate cartilage degeneration. Apparent matrix degradation was observable in the deep zone of cartilage even in the early stages of OA. FT-IRIS studies also found that within the territorial matrix of the cartilage cells (chondrocytes), proteoglycan content increased with progression of cartilage degeneration while the collagen content remained the same, but the collagen integrity decreased. Regenerative (repair) tissue from microfracture treatment of an equine cartilage defect showed significant changes in collagen distribution and loss in proteoglycan content compared to the adjacent normal cartilage, with collagen fibrils demonstrating a random orientation in most of the repair tissue. These studies demonstrate that FT-IRIS is a powerful technique that can provide detailed ultrastructural information on heterogeneous tissues such as diseased cartilage and thus has great potential as a diagnostic modality for cartilage degradation and repair.

Histological and molecular structure characterization of annular collagen after intradiskal electrothermal annuloplasty.

The mechanism of pain relief of intradiskal electrothermal annuloplasty (IDET) in the treatment of lumbar diskogenic pain is uncertain. Theories include sealing of annular fissures via collagen denaturation and contraction. Prior studies offer conflicting qualitative data on the ability of IDET to denature collagen. The objective of the present study is to evaluate IDET treatment effect on annular collagen using quantitative data supplied by Fourier-transform infrared imaging spectroscopy. The posterior annulus of disks (n = 3) from an intact human cadaveric spine at room temperature were treated with two different radiothermal catheters using standard intradiskal electrothermal annuloplasty (IDET) heating protocols. Disks were dissected free with catheters in place and fixed in formalin. Channels created by the catheters were marked and catheters were removed. Tissue samples of treated areas adjacent to the channels and internal control areas from the same disk were stained for light microscopy and placed on barium sulfate windows for Fourier transform infrared imaging spectroscopy (FT-IRIS) analysis. Treated areas showed evidence of disruption in the fibrillar organization of annular collagen by light microscopy compared to intact stroma from control areas. Quantitative FT-IRIS analysis compared ratios of wavenumber regions known to be sensitive to collagen denaturation. Mean values for the ratios amide II/1,338 cm(-1) (137.21 +/- 25.84 treated, 76.94 +/- 16.77 control) and 1,640/1,660 cm(-1) (0.98 +/- 0.03 treated, 0.89 +/- 0.03 control) were significantly different between treated and control samples (p < 0.001), indicating a breakdown in collagen integrity. Separate analysis by catheter type suggests that catheter design may impact treatment effect.

Statistical comparison of Fourier transform infrared spectra.

Spectroscopic assessment of whether a biological sample has changed as a result of processing or degradation is generally carried out by qualitative comparison of spectra, without statistical analysis, resulting in a subjective evaluation of sample stability. Here, we present a formalism for quantitative statistical comparison of signal-averaged Fourier transform infrared spectra, commonly used to assess molecular properties of biological samples. Expressions are derived permitting the comparison of 1. single beam spectra; 2. transmittance spectra obtained by calculating the ratio of single beam spectra of a sample and background; and 3. absorbance spectra derived from transmittance spectra. An application of these results to the degradation of cartilage is presented. Two absorbance spectra of a cartilage sample taken in succession are found to be statistically identical with respect to the ratio of the amplitude of the amide I band to the amplitude of the amide II band. However, a spectrum of the same sample acquired after a 24-h degradation period, while similar to the spectrum of the fresh sample, is found to have an altered ratio of these spectral band amplitudes, consistent with degradation of the cartilage matrix.