Biomedical subjects

David H Kohn

Publications and source records attributed to David H Kohn.

9 recordsLinked to original sources

PubMed2006

Is photobleaching necessary for Raman imaging of bone tissue using a green laser?

Raman microspectroscopy is widely used for musculoskeletal tissues studies. But the fluorescence background obscures prominent Raman bands of mineral and matrix components of bone tissue. A 532-nm laser irradiation has been used efficiently to remove the fluorescence background from Raman spectra of cortical bone. Photochemical bleaching reduces over 80% of the fluorescence background after 2 h and is found to be nondestructive within 40 min. The use of electron multiplying couple charge detector (EMCCD) enables to acquire Raman spectra of bone tissues within 1-5 s range and to obtain Raman images less than in 10 min.

Animals↗

PubMed2006

The mechanical phenotype of biglycan-deficient mice is bone- and gender-specific.

Biglycan (bgn) is a small leucine-rich proteoglycan (SLRP) enriched in the extracellular matrix of skeletal tissues. While bgn is known to be involved in the growth and differentiation of osteoblast precursor cells and regulation of collagen fibril formation, it is unclear how these functions impact bone's geometric and mechanical properties, properties which are integral to the structural function of bone. Because the genetic control of bone structure and function is both local- and gender-specific and because there is evidence of gender-specific effects associated with genetic deficiencies, it was hypothesized that the engineered deletion of the gene encoding bgn would result in a cortical bone mechanical phenotype that was bone- and gender-specific. In 11-week-old C57BL6/129 mice, the cortical bone in the mid-diaphyses of the femora and tibiae of both genders was examined. Phenotypic changes in bgn-deficient mice relative to wild type controls were assayed by four-point bending tests to determine mechanical properties at the whole bone (structural) and tissue levels, as well as analyses of bone geometry and bone formation using histomorphometry. Of the bones examined, bgn deficiency most strongly affected the male tibiae, where enhanced cross-sectional geometric properties and bone mineral density were accompanied by decreased tissue-level yield strength and pre-yield structural deformation and energy dissipation. Because pre-yield properties alone were impacted, this implies that the gene deletion causes important alterations in mineral and/or the matrix/mineral ultrastructure and suggests a new understanding of the functional role of bgn in regulating bone mineralization in vivo.

Animals↗

PubMed2006

Three structural roles for water in bone observed by solid-state NMR.

Hydrogen-bearing species in the bone mineral environment were investigated using solid-state NMR spectroscopy of powdered bone, deproteinated bone, and B-type carbonated apatite. Using magic-angle spinning and cross-polarization techniques three types of structurally-bound water were observed in these materials. Two of these water types occupy vacancies within the apatitic mineral crystal in synthetic carbonated apatite and deproteinated bone and serve to stabilize these defect-containing crystals. The third water was observed at the mineral surface in unmodified bone but not in deproteinated bone, suggesting a role for this water in mediating mineral-organic matrix interactions. Direct evidence of monohydrogen phosphate in a (1)H NMR spectrum of unmodified bone is presented for the first time. We obtained clear evidence for the presence of hydroxide ion in deproteinated bone by (1)H MAS NMR. A (1)H-(31)P heteronuclear correlation experiment provided unambiguous evidence for hydroxide ion in unmodified bone as well. Hydroxide ion in both unmodified and deproteinated bone mineral was found to participate in hydrogen bonding with neighboring water molecules and ions. In unmodified bone mineral hydroxide ion was found, through a (1)H-(31)P heteronuclear correlation experiment, to be confined to a small portion of the mineral crystal, probably the internal portion.

Animals↗

PubMed2006

Substrate mineralization stimulates focal adhesion contact redistribution and cell motility of bone marrow stromal cells.

Understanding the mechanisms of substrate based control of cell function is critical to the design of biomaterials. Cells interact with their extracellular matrix through cell adhesion contacts. We have previously described the self assembly of bone-like mineral onto an organic template and have shown that these biomimetic surfaces lead to an increased volume fraction of bone regenerated in vivo. In the present study, we compared the distribution of cell adhesion contacts, cell spreading, and cell motility of murine bone marrow stromal cells (BMSC) on mineralized vs. nonmineralized substrates. We developed a new approach for quantification of cell-material interactions and demonstrated that cell adhesion contacts on mineralized substrates were distributed throughout the cell surface contacting the substrate, whereas on nonmineralized substrates cell adhesion contacts were present near the cell periphery. We propose that mineralized substrates stimulate the predominant expression of fibrillar contacts, and nonmineralized substrates stimulate expression of focal adhesion contacts. Cell motility assays with colloidal gold demonstrated a statistically significant decrease in the average phagokinetic index of migrating cells on mineralized vs. nonmineralized substrates after 90 min of cell seeding. We propose that the physical-chemical properties of the substrate, altered by mineralization, cause expression of specific types of cell contacts and, as a result, modify molecular mechanisms responsible for cell spreading, motility, and possibly differentiation.

Animals↗

PubMed2005

Micro- and nano-structural analyses of damage in bone.

Skeletal fractures represent a significant medical and economic burden for our society. In the US alone, age-related hip, spine, and wrist fractures accounted for more than $17 billion in direct health care costs in 2001. Moreover, skeletal fractures are not limited to the elderly; stress fractures and impact/trauma-related fractures are a significant problem in younger people also. Gaining insight into the mechanisms of fracture and how these mechanisms are modulated by intrinsic as well as extrinsic factors may improve the ability to define fracture risk and develop and assess preventative therapies for skeletal fractures. Insight into failure mechanisms of bone, particularly at the ultrastructural-level, is facilitated by the development of improved means of defining and measuring tissue quality. Included in these means are microscopic and spectroscopic techniques for the direct observation of crack initiation, crack propagation, and fracture behavior. In this review, we discuss microscopic and spectroscopic techniques, including laser scanning confocal microscopy (LSCM), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Raman spectroscopic imaging for visually observing microdamage in bone, and the current understanding of damage mechanisms derived from these techniques.

Animals↗

PubMed2005

Spatial control of protein within biomimetically nucleated mineral.

An ideal approach for bone tissue engineering allows for osteoconductivity, osteoinductivity, and cell transplantation. In this study, we examined coprecipitation and surface adsorption schemes with respect to their abilities to control the spatial quantity and localization of a model protein, bovine serum albumin (BSA), that is incorporated into a biomimetic apatite layer nucleated onto polylactic-co-glycolic acid (PLGA) films. Protein incorporation was characterized by determining protein: presence, quantity loaded, retention, effects on mineral morphology, and localization. FT-IR confirmed the presence of protein in all coprecipitation samples with stronger peaks in the coprecipitated samples compared to the surface adsorbed samples. Coprecipitation resulted in higher loading capacities and higher protein retention versus adsorption. Protein incorporation via coprecipitation changed the mineral morphology from sharp plate-like structures to more rounded structures, whereas, surface adsorption did not change mineral structure. By using confocal microscopy to examine the incorporation of fluorescently labeled proteins, spatial control over protein localization was exhibited. By controlling the loading quantity and localization of the model protein through the mineral thickness, a desired release profile can be achieved. A desired and effective delivery system of biological agents utilizing coprecipitation for bone regeneration can therefore be designed.

Adsorption↗

PubMed2004

Highly ordered interstitial water observed in bone by nuclear magnetic resonance.

UNLABELLED: NMR was used to study the nanostructure of bone tissue. Distance measurements show that the first water layer at the surface of the mineral in cortical bone is structured. This water may serve to couple the mineral to the organic matrix and may play a role in deformation. INTRODUCTION: The unique mechanical characteristics of bone tissue have not yet been satisfactorily connected to the exact molecular architecture of this complex composite material. Recently developed solid-state nuclear magnetic resonance (NMR) techniques are applied here to the mineral component to provide new structural distance constraints at the subnanometer scale. MATERIALS AND METHODS: NMR dipolar couplings between structural protons (OH(-) and H(2)O) and phosphorus (PO(4)) or carbon (CO(3)) were measured using the 2D Lee-Goldburg Cross-Polarization under Magic-Angle Spinning (2D LG-CPMAS) pulse sequence, which simultaneously suppresses the much stronger proton-proton dipolar interactions. The NMR dipolar couplings measured provide accurate distances between atoms, e.g., OH and PO(4) in apatites. Excised and powdered femoral cortical bone was used for these experiments. Synthetic carbonate ( approximately 2-4 wt%)-substituted hydroxyapatite was also studied for structural comparison. RESULTS: In synthetic apatite, the hydroxide ions are strongly hydrogen bonded to adjacent carbonate or phosphate ions, with hydrogen bond (O-H) distances of approximately 1.96 A observed. The bone tissue sample, in contrast, shows little evidence of ordered hydroxide. Instead, a very ordered (structural) layer of water molecules is identified, which hydrates the small bioapatite crystallites through very close arrangements. Water protons are approximately 2.3-2.55 A from surface phosphorus atoms. CONCLUSIONS: In synthetic carbonated apatite, strong hydrogen bonds were observed between the hydroxide ions and structural phosphate and carbonate units in the apatite crystal lattice. These hydrogen bonding interactions may contribute to the long-range stability of this mineral structure. The biological apatite in cortical bone tissue shows evidence of hydrogen bonding with an ordered surface water layer at the faces of the mineral particles. This structural water layer has been inferred, but direct spectroscopic evidence of this interstitial water is given here. An ordered structural water layer sandwiched between the mineral and the organic collagen fibers may affect the biomechanical properties of this complex composite material.

Animals↗

PubMed2004

Bone tissue ultrastructural response to elastic deformation probed by Raman spectroscopy.

Raman spectroscopy is used as a probe of ultrastructural (molecular) changes in both the mineral and matrix (protein and glycoprotein, predominantly type I collagen) components in real time of murine cortical bone as it responds to elastic deformation. Because bone is ia composite material, its mechanical properties are dependent on the structure and composition at a variety of dimensional scales. At the ultrastructural level, crystal structure and protein secondary structure distort as the tissue is loaded. These structural changes are followed as perturbations to tissue spectra. We load murine femora in a custom-made mechanical tester that fits on the stage of a Raman microprobe and can accept hydrated tissue specimens. As the specimen is loaded in tension, the shifts in mineral P-O4 v1 are followed with the microprobe. Average load and strain are measured using a load cell. These devices ensure that specimens are not loaded to or beyond the yield point. Changes occur in the mineral component of bone as a response to loading in the elastic regime. We propose that the mineral apatitic crystal lattice is deformed by movement of calcium and other ions. Raman microspectroscopy shows that bone mineral is not a passive contributor to tissue strength. The mineral active response to loading may function as a local energy storage and dissipation mechanism, thus helping to protect tissue from catastrophic damage.

Algorithms↗

PubMed2002

Effects of pH on human bone marrow stromal cells in vitro: implications for tissue engineering of bone.

The objective of this study was to address the hypothesis that changes in extracellular pH alter collagen gene expression, collagen synthesis, and alkaline phosphatase activity in bone marrow stromal cells (BMSCs). Potential effects of pH on cell function are of particular importance for tissue engineering because considerable effort is being placed on engineering biodegradable polymers that may generate a local acidic microenvironment on degradation. Human and murine single-cell marrow suspensions were plated at a density of 2 x 10(4) cells/cm(2). After 7 days in culture, the pH of the culture medium was adjusted to one of six ranges: > or = 7.8, 7.5.-7.7, 7.2-7.4, 6.9-7.1, 6.6-6.8, or < or = 6.5. After 48 h of exposure to an altered pH, alkaline phosphatase activity and collagen synthesis decreased significantly with decreasing pH. This decrease was two-to threefold as pH decreased from 7.5 to 6.6. In contrast, alpha1(I) procollagen mRNA levels increased two- to threefold as pH was decreased. The trend in osteocalcin mRNA expression was opposite to that of collagen. Small shifts in extracellular pH led to significant changes in the ability of BMSCs to express markers of the osteoblast phenotype. These pH effects potentially relate to the microenvironment supplied by a tissue-engineering scaffold and suggest that degrading polymer scaffolds may influence the biologic activity of the cells in the immediate environment.

Alkaline Phosphatase↗