Retention of Endogenous Viable Cells Enhances the Anti-Inflammatory Activity of Cryopreserved Amnion.

Objective: Human amniotic membrane (hAM) has been used to treat wounds for more than 100 years. However, widespread use of fresh hAM has been limited due to its short shelf life and safety concerns. To overcome these concerns, different preservation methods have been introduced. The majority of these methods result in devitalized hAM (dev-hAM). Recently, we developed a cryopreservation method that retains all hAM components intact (int-hAM), including viable endogenous cells. To understand the advantages of retaining viable cells in preserved hAM, we compared the anti-inflammatory properties of int-hAM and dev-hAM. Approach: The tissue composition of int-hAM and dev-hAM was compared with fresh hAM through histology and cell viability analysis. We also evaluated the ability of int-hAM and dev-hAM to regulate tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), and IL-10 release when co-cultured with immune cells; to produce prostaglandin E2 (PGE2) on TNF-α stimulation; and to inhibit proteases. Results: Int-hAM maintained the structural and cellular integrity of fresh hAM. Int-hAM had >80% cell viability post-thaw and remained viable for at least a week in culture. Viable cells were not detected in dev-hAM. Compared with dev-hAM, int-hAM showed significantly greater downregulation of TNF-α and IL-1α, upregulation of PGE2 and IL-10, and stronger inhibition of collagenase. Innovation and Conclusion: A new cryopreservation method has been developed to retain all native components of hAM. For the first time, we show that viable endogenous cells significantly augment the anti-inflammatory activity of cryopreserved hAM.

Angiogenic Potential of Cryopreserved Amniotic Membrane Is Enhanced Through Retention of All Tissue Components in Their Native State.

Objective: Chronic wounds have inadequate microvasculature (or blood vessels), resulting in poor healing. Both fresh human amniotic membrane (hAM) containing viable cells and devitalized hAM have been shown to stimulate angiogenesis in chronic wounds. However, the importance of retaining viable endogenous cells on the angiogenic activity of hAM remains unknown. To understand their role, we compared the angiogenic potential of intact cryopreserved hAM containing viable cells (int-hAM) with devitalized cryopreserved hAM (dev-hAM). Approach: The effects of conditioned medium (CM) derived from int-hAM and dev-hAM on endothelial cell migration and tube formation were compared. Int-hAM and dev-hAM CM and tissues were tested for key angiogenic factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor-BB (PDGF-BB) after 7 days in culture. The role of VEGF in int-hAM-mediated tube formation was analyzed through inhibition of its activity by anti-VEGF antibody. Results: CM from int-hAM showed greater endothelial cell recruitment and tube formation compared with dev-hAM. Significantly higher levels of VEGF were detected in int-hAM CM after 1 week compared with dev-hAM CM. Int-hAM tissue also had significantly greater expression of VEGF and bFGF relative to dev-hAM. A similar trend was observed for PDGF-BB. Neutralization of VEGF in int-hAM CM significantly inhibited tube formation compared with int-hAM CM alone. Innovation and Conclusion: Preservation of all native hAM components, including viable endogenous cells, enhances the angiogenic effect of cryopreserved hAM. This effect is mediated through higher levels of angiogenic factors, especially VEGF, produced by int-hAM.

Alendronate treatment of the brtl osteogenesis imperfecta mouse improves femoral geometry and load response before fracture but decreases predicted material properties and has detrimental effects on osteoblasts and bone formation.

Long courses of bisphosphonates are widely administered to children with osteogenesis imperfecta (OI), although bisphosphonates do not block mutant collagen secretion and may affect bone matrix composition or structure. The Brtl mouse has a glycine substitution in col1a1 and is ideal for modeling the effects of bisphosphonate in classical OI. We treated Brtl and wildtype mice with alendronate (Aln; 0.219 mg/kg/wk, SC) for 6 or 12 wk and compared treated and untreated femora of both genotypes. Mutant and wildtype bone had similar responses to Aln treatment. Femoral areal BMD and cortical volumetric BMD increased significantly after 12 wk, but femoral length and growth curves were unaltered. Aln improved Brtl diaphyseal cortical thickness and trabecular number after 6 wk and cross-sectional shape after 12 wk. Mechanically, Aln significantly increased stiffness in wildtype femora and load to fracture in both genotypes after 12 wk. However, predicted material strength and elastic modulus were negatively impacted by 12 wk of Aln in both genotypes, and metaphyseal remnants of mineralized cartilage also increased. Brtl femoral brittleness was unimproved. Brtl osteoclast and osteoblast surface were unchanged by treatment. However, decreased mineral apposition rate and bone formation rate/bone surface and the flattened morphology of Brtl osteoblasts suggested that Aln impaired osteoblast function and matrix synthesis. We conclude that Aln treatment improves Brtl femoral geometry and load to fracture but decreases bone matrix synthesis and predicted material modulus and strength, with striking retention of mineralized cartilage. Beneficial and detrimental changes appear concomitantly. Limiting cumulative bisphosphonate exposure of OI bone will minimize detrimental effects.

Cellular mechanism of decreased bone in Brtl mouse model of OI: imbalance of decreased osteoblast function and increased osteoclasts and their precursors.

The Brtl mouse, a knock-in model for moderately severe osteogenesis imperfecta (OI), has a G349C substitution in half of type I collagen alpha1(I) chains. We studied the cellular contribution to Brtl bone properties. Brtl cortical and trabecular bone are reduced before and after puberty, with BV/TV decreased 40-45%. Brtl ObS/BS is comparable to wildtype, and Brtl and wildtype marrow generate equivalent number of colony-forming units (CFUs) at both ages. However, OcS/BS is increased in Brtl at both ages (36-45%), as are TRACP(+) cell numbers (57-47%). After puberty, Brtl ObS/BS decreases comparably to wildtype mice, but osteoblast matrix production (MAR) decreases to one half of wildtype values. In contrast, Brtl OcS falls only moderately (approximately 16%), and Brtl TRACP staining remains significantly elevated compared with wildtype. Consequently, Brtl BFR decreases from normal at 2 mo to one half of wildtype values at 6 mo. Immunohistochemistry and real-time RT-PCR show increased RANK, RANKL, and osteoprotegerin (OPG) levels in Brtl, although a normal RANKL/OPG ratio is maintained. TRACP(+) precursors are markedly elevated in Brtl marrow cultures and form more osteoclasts, suggesting that osteoclast increases arise from more RANK-expressing precursors. We conclude that osteoblasts and osteoclasts are unsynchronized in Brtl bone. This cellular imbalance results in declining BFR as Brtl ages, consistent with reduced femoral geometry. The disparity in cellular number and function results from poorly functioning osteoblasts in addition to increased RANK-expressing precursors that respond to normal RANKL/OPG ratios to generate more bone-resorbing osteoclasts. Interruption of the stimulus that increases osteoclast precursors may lead to novel OI therapies.

Deficiency of cartilage-associated protein in recessive lethal osteogenesis imperfecta.

Classic osteogenesis imperfecta, an autosomal dominant disorder associated with osteoporosis and bone fragility, is caused by mutations in the genes for type I collagen. A recessive form of the disorder has long been suspected. Since the loss of cartilage-associated protein (CRTAP), which is required for post-translational prolyl 3-hydroxylation of collagen, causes severe osteoporosis in mice, we investigated whether CRTAP deficiency is associated with recessive osteogenesis imperfecta. Three of 10 children with lethal or severe osteogenesis imperfecta, who did not have a primary collagen defect yet had excess post-translational modification of collagen, were found to have a recessive condition resulting in CRTAP deficiency, suggesting that prolyl 3-hydroxylation of type I collagen is important for bone formation.

Defective osteogenesis of the stromal stem cells predisposes CD18-null mice to osteoporosis.

Osteogenesis by the bone marrow stromal stem cells (BMSSCs) supports continuous bone formation and the homeostasis of the bone marrow microenvironment. The mechanism that controls the proliferation and differentiation of BMSSCs is not fully understood. Here, we report that CD18, a surface protein present primarily on hematopoietic cells, but not on differentiated mesenchymal cells, is expressed by the stromal stem cells and plays a critical role in the osteogenic process. Constitutive expression of CD18 on BMSSCs using a retroviral promoter significantly enhances bone formation in vivo, whereas genetic inactivation of CD18 in mice leads to defective osteogenesis due to decreased expression of the osteogenic master regulator Runx2/Cbfa1. The defective osteogenesis of the CD18-null BMSSCs can be restored by expressing full-length, but not cytoplasmic domain-truncated, CD18. Radiographic analyses with dual-energy x-ray absorptiometry and 3D microcomputed tomography show that mice lacking CD18 have decreased bone mineral density and exhibit certain features of osteoporosis. Altogether, this work demonstrates that CD18 functions critically in the osteogenesis of BMSSCs, and thus lack of CD18 expression in the leukocyte adhesion deficiency patients may predispose them to osteoporosis.

Brittle IV mouse model for osteogenesis imperfecta IV demonstrates postpubertal adaptations to improve whole bone strength.

UNLABELLED: The Brtl mouse model for type IV osteogenesis imperfecta improves its whole bone strength and stiffness between 2 and 6 months of age. This adaptation is accomplished without a corresponding improvement in geometric resistance to bending, suggesting an improvement in matrix material properties. INTRODUCTION: The Brittle IV (Brtl) mouse was developed as a knock-in model for osteogenesis imperfecta (OI) type IV. A Gly349Cys substitution was introduced into one col1a1 allele, resulting in a phenotype representative of the disease. In this study, we investigate the effect of the Brtl mutation on whole bone architecture, strength, and composition across a range of age groups. MATERIALS AND METHODS: One-, 2-, 6-, and 12-month-old Brtl and wildtype (WT) mice were analyzed. Femurs were assessed at the central diaphysis for cortical geometric parameters using microCT and were subsequently mechanically tested to failure by four-point bending. Matrix material properties were predicted using microCT data to normalize data from mechanical tests. Raman spectroscopy and DXA were used to assess matrix composition. RESULTS: Our findings show a postpubertal adaptation in which Brtl femoral strength and stiffness increase through a mechanism independent of changes in whole bone geometry. These findings suggest an improvement in the material properties of the bone matrix itself, rather than improvements in whole bone geometry, as seen in previous mouse models of OI. Raman spectroscopic results suggest these findings may be caused by changes in mineral/matrix balance rather than improvements in mineral crystallinity. CONCLUSIONS: Our findings parallel the currently unexplained clinical observation of decreased fractures in human OI patients after puberty. The Brtl mouse remains an important tool for investigating therapeutic interventions for OI.

Intron 1 is required for cell type-specific, but not injury-responsive, peripherin gene expression.

The "primitive" neurons of the peripheral nervous system (PNS) have the remarkable ability to regenerate new fibers. This regenerative process requires a sequence of gene activation and repression that is poorly understood. One gene that is almost exclusively expressed in neurons of the PNS and is activated after nerve injury is the peripherin intermediate filament gene, but little is known about the genomic elements that control either its restricted expression or its response to nerve injury in adult mice. Previous studies suggested that both 5' flanking sequence and intragenic regions were required for cell type-specific and injury-specific expression. To determine which intragenic regions were critical, mice were generated that expressed peripherin transgenes lacking different introns. Analyses of these mice revealed that deletion of introns 2-8 had no effect on either the cell type-specific or injury-specific expression of the peripherin gene; however, the remaining intron, intron 1, differentially bound Sp1 transcription-related proteins/protein complexes in extracts from peripherin-expressing and nonexpressing tissues. Furthermore, a transgene that lacked intron 1 was not expressed in many neurons that contain endogenous peripherin but was activated after injury. Thus, accurate cell type-specific peripherin gene expression in the PNS depends on elements within intron 1, but other sequences, most likely in the 5'flanking region, are required for activating the peripherin gene in response to nerve injury.