Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves 129-157). Also available on the Internet.

Cell-derived (CDM) matrix scaffolds provide a 3-dimensional (3D) matrix material that recapitulates a native, human extracellular matrix (ECM) microenvironment. CDMs are a heterogeneous source of ECM proteins with a composition dependent on the cell source and its phenotype. CDMs have several applications, such as for development of cell culture substrates to study stromal cell propagation and differentiation, as well as cell or drug delivery vehicles, or for regenerative biomaterial applications. Although CDMs are versatile and exhibit advantageous structure and activity, their use has been hindered due to the prolonged culture time required for ECM deposition and maturation in vitro. Macromolecular crowding (MMC) has been shown to increase ECM deposition and organization by limiting the diffusion of ECM precursor proteins and allowing the accumulation of matrix at the cell layer. A commonly used crowder that has been shown to increase ECM deposition in vitro is Ficoll, and was used in this study as a positive control to assess matrix deposition. Hyaluronic acid (HA), a natural crowding macromolecule expressed at high levels during fetal development, has been shown to play a role in ECM production, organization, and assembly in vivo. HA has not been investigated as a crowding molecule for matrix deposition or development of CDMs in vitro. This dissertation focused on 2 aims supporting the development of a functional, human dermal fibroblast-derived ECM material for the delivery deliver an antimicrobial peptide, cCBD-LL37, and for potentially promoting a pro-angiogenic environment. The goal of this thesis was to evaluate the effects of high molecular weight (HMW) HA as a macromolecular crowding agent on in vitro deposition of ECM proteins important for tissue regeneration and angiogenesis. A pilot proteomics study supported the use of HA as a crowder, as it preliminarily showed increases in ECM proteins and increased retention of ECM precursor proteins at the cell layer; thus supporting the use of HA as a crowder molecule. In the presence of HA, human dermal fibroblasts demonstrated an increase in ECM deposition comparable to the effects of Ficoll 70/400 at day 3 using Raman microspectroscopy. It was hypothesized that HA promotes matrix deposition through changes on ECM gene expression. However, qRT-PCR results indicate that HA and Ficoll 70/400 did not have a direct effect on collagen gene expression, but differences in matrix crosslinking and proteinase genes were observed. Decellularized CDMs were then used to assess CDM stiffness and endothelial sprouting, which indicated differences in structural organization of collagen, and preliminarily suggests that there are differences in endothelial cell migration depending on the crowder agent used in culture. Finally, the collagen retained in the decellularized CDM matrix prepared under MMC supported the binding of cCBD-LL37 with retention of antimicrobial activity when tested against E.coli. Overall, the differences in matrix deposition profiles in HA versus Ficoll crowded cultures may be attributed to crowder molecule-mediated differences in matrix crosslinking, turnover, and organization as indicated by differences in collagen deposition, matrix metalloproteinase expression, and matrix stiffness. MMC is a valuable tool for increasing matrix deposition, and can be combined with other techniques, such as low oxygen and bioreactor cultures, to promote development of a biomanufactured CDM-ECM biomaterial. Successful development of scalable CDM materials that stimulate angiogenesis and support antimicrobial peptide delivery would fill an important unmet need in the treatment of non-healing, chronic, infected wounds.

Cell-derived (CDM) matrix scaffolds provide a 3-dimensional (3D) matrix material that recapitulates a native, human extracellular matrix (ECM) microenvironment. CDMs are a heterogeneous source of ECM proteins with a composition dependent on the cell source and its phenotype. CDMs have several applications, such as for development of cell culture substrates to study stromal cell propagation and differentiation, as well as cell or drug delivery vehicles, or for regenerative biomaterial applications. Although CDMs are versatile and exhibit advantageous structure and activity, their use has been hindered due to the prolonged culture time required for ECM deposition and maturation in vitro. Macromolecular crowding (MMC) has been shown to increase ECM deposition and organization by limiting the diffusion of ECM precursor proteins and allowing the accumulation of matrix at the cell layer. A commonly used crowder that has been shown to increase ECM deposition in vitro is Ficoll, and was used in this study as a positive control to assess matrix deposition. Hyaluronic acid (HA), a natural crowding macromolecule expressed at high levels during fetal development, has been shown to play a role in ECM production, organization, and assembly in vivo. HA has not been investigated as a crowding molecule for matrix deposition or development of CDMs in vitro. This dissertation focused on 2 aims supporting the development of a functional, human dermal fibroblast-derived ECM material for the delivery deliver an antimicrobial peptide, cCBD-LL37, and for potentially promoting a pro-angiogenic environment. The goal of this thesis was to evaluate the effects of high molecular weight (HMW) HA as a macromolecular crowding agent on in vitro deposition of ECM proteins important for tissue regeneration and angiogenesis. A pilot proteomics study supported the use of HA as a crowder, as it preliminarily showed increases in ECM proteins and increased retention of ECM precursor proteins at the cell layer; thus supporting the use of HA as a crowder molecule. In the presence of HA, human dermal fibroblasts demonstrated an increase in ECM deposition comparable to the effects of Ficoll 70/400 at day 3 using Raman microspectroscopy. It was hypothesized that HA promotes matrix deposition through changes on ECM gene expression. However, qRT-PCR results indicate that HA and Ficoll 70/400 did not have a direct effect on collagen gene expression, but differences in matrix crosslinking and proteinase genes were observed. Decellularized CDMs were then used to assess CDM stiffness and endothelial sprouting, which indicated differences in structural organization of collagen, and preliminarily suggests that there are differences in endothelial cell migration depending on the crowder agent used in culture. Finally, the collagen retained in the decellularized CDM matrix prepared under MMC supported the binding of cCBD-LL37 with retention of antimicrobial activity when tested against E.coli. Overall, the differences in matrix deposition profiles in HA versus Ficoll crowded cultures may be attributed to crowder molecule-mediated differences in matrix crosslinking, turnover, and organization as indicated by differences in collagen deposition, matrix metalloproteinase expression, and matrix stiffness. MMC is a valuable tool for increasing matrix deposition, and can be combined with other techniques, such as low oxygen and bioreactor cultures, to promote development of a biomanufactured CDM-ECM biomaterial. Successful development of scalable CDM materials that stimulate angiogenesis and support antimicrobial peptide delivery would fill an important unmet need in the treatment of non-healing, chronic, infected wounds.

No description available.

published_or_final_version / Orthopaedics and Traumatology / Master / Master of Philosophy

published_or_final_version / Mechanical Engineering / Master / Master of Philosophy

Traumatic skeletal muscle injuries that include loss of large amounts of muscle mass are becoming more common in today’s warfare. Traditional treatments often do not prevent long term functional impairments. Using a decellularized extracellular matrix (ECM) as scaffolding to replace lost muscle tissue allows for transmission of force through the injury site, and provides a suitable microenvironment receptive to myofiber growth. Seeding the ECM with progenitor cells improves cellular content in the defect area. Exercise exposes the muscle to improved blood flow as well as higher than normal loading. This results in increased blood vessel density as well as higher levels of cellular content, and near complete restoration of function. / text

Cardiovascular disease, often induced by hypertension, represents a serious health threat, is a primary cause of death worldwide, and results in altered cardiovascular function and ECM composition. Hypertension and related cardiovascular diseases are associated with immune dysfunction. This dissertation investigated the role of T-lymphocytes in modulating cardiovascular function and ECM composition as a possible therapeutic for the treatment of cardiovascular disease. Study one investigated the role of TCR peptide in the development of hypertension and subsequent cardiovascular changes in Balb/C mice. The coadminstration of TCR and L-NAME/8% NaCl reduced the effects of L-NAME/8% NaCl, decreasing blood pressure and crosslinked collagen compared to L-NAME/8% NaCl alone. Study two examined the effects of T-lymphocyte function on cardiovascular structure and function. Adoptive transfer of T-lymphocytes from C57BL/6 WT mice into C57BL/6 SCID mice induced changes in the SCID so that it resembled the WT donor, with increased percent crosslinked collagen and LOX activity. Hemodynamics in the SCID recipient resembled that of the WT and were significantly different from the sham injected SCID. Study three combined aspects of both previous studies. T-lymphocytes were adoptively transferred from hypertensive WT donors into naïve SCID recipients, who developed hypertension and cardiovascular function resembling the hypertensive donor, as well as changes in the ECM, including increased collagen crosslinking. Study four investigated the effect of strain specific T-lymphocyte polarization on hypertension induced cardiac ECM remodeling. Balb/C, C57BL/6 WT, and C57BL/6 SCID had divergent responses to L-NAME induced hypertension. Ventricular stiffness increased in Balb/C, decreased in C57 SCID and did not change in C57 WT; LOX activity changed correspondingly in all groups. The final study examined the effect of TCR administration on LOX activity and collagen crosslinking. Th1 polarization increased LOX activity and crosslinked collagen with corresponding changes in cardiovascular function. In conclusion, modulation of T-lymphocyte function alters cardiovascular function and ECM composition in pathologic and non-pathologic conditions. Immune modulation should be further investigated as a therapeutic for cardiovascular disease.

Millions of people around the world suffer from the debilitating effects of inflammatory bone diseases characterized by excessive bone loss due to an increase in osteoclast formation and activity. Osteoclasts are multinucleated cells responsible for bone resorption in health and disease. Arthritic joints also have elevated levels of extracellular matrix proteins affecting the disease progression. The interaction between osteoclasts and the external milieu comprised of extracellular matrix proteins through integrins is essential for modulating the formation and activity of osteoclasts. The focus of this thesis was to elucidate how the interaction between the extracellular matrix proteins and osteoclasts regulates osteoclast formation and activity and the role of alphavbeta3 in this process. In primary rabbit osteoclast cultures, blocking the integrin alphavbeta3 using Vitaxin, an anti-human alphavbeta3 antibody, decreased osteoclast resorption by decreasing osteoclast attachment. Vitaxin’s inhibitory effect on osteoclast attachment was enhanced when osteoclasts were pretreated with M-CSF, a growth factor known to induce an activated conformation of the integrin alphavbeta3. Using the RAW264.7 cell line, the effects of the matrix proteins fibronectin and vitronectin on osteoclast activity were compared to those of osteopontin. Both fibronectin and vitronectin decreased the number of osteoclasts formed compared to osteopontin. Fibronectin’s effect on osteoclastogenesis was through decreasing pre-osteoclast migration and/or fusion but not through inhibiting their recruitment. In contrast, fibronectin induced resorption through increasing resorptive activity per osteoclast in comparison to vitronectin and osteopontin. These stimulatory effects were accompanied by an increase in the pro-inflammatory cytokines nitric oxide and IL-1beta Crosstalk between the signalling pathways of nitric oxide and IL-1betawas suggested by the ability of the nitric oxide inhibitor to decrease the level of IL-1beta which occurred exclusively on fibronectin. Osteoclasts on fibronectin also had a compact morphology with the smallest planar area while vitronectin increased the percentage of osteoclast with migratory morphology and osteopontin induced osteoclast spreading. The increase in compact morphology on fibronectin was associated with a decrease in extracellular pH. Low extracellular pH was found to increase the total time osteoclasts spend in a compact phase. These results show that matrix proteins differentially regulate osteoclast formation, activity and morphology.

Hyaluronic acid (HA) is one of the major components of the extracellular matrix; and may exhibit different biological functions, dependent on polymer molecular weight (MW). The signalling events performed by HA are mediated through interactions with its main cell receptors: CD44 and RHAMM. However, the direct effect between the HA MW and the expression of CD44 and RHAMM remains unclear. This study aimed to investigate whether different HA polymer MW alters the proliferation of tumour-derived cell lines, and whether different HA-sized has an effect on the regulation of the expression of CD44 and RHAMM. In order to determine size-specific responses of tumour cells of defined fragment MW, this investigation was undertaken using HA-tethered culture surfaces. Four surfaces were constructed, coated with polymers of different MWs. HA (4, 234, 2590 kDa) and an oligomer mixture were tethered onto an aminosilane (AHAPTMS)-treated glass surfaces using a carbodiimide reaction. Surfaces were analysed using a toolbox of in situ characterisation techniques, including wettability measurements, QCM, AFM and confocal microscopy. Using the constructed surfaces was demonstrated that HA-polymer MW modulates cell proliferation of human bladder (RT112 and T24) and prostate (PC3 and PNT1A) cell lines, with low HA MW (HA4) increasing proliferation, whereas a decrease is seen in the presence of medium (HA234) and high MW fragments (HA2590). The proliferation stimulus performed by HA was found to be phenotype dependent, with HA4 surfaces stimulating an increased proliferation in those less invasive cell lines (T24 and PNT1A), while HA234 and HA2590 inducing a sharper decrease in the most malignant tumour cell lines (RT112 and PC3). It was also demonstrated that the regulation of CD44 and RHAMM transcripts expression appears to be phenotype dependent but not HA-MW dependent. HA down-regulates CD44 and RHAMM in the most malignant cell lines; with up-regulation of the expression of the cell receptors in the less invasive cell lines. In addition, the presence of exogenous HA was shown to be involved in the regulation of the expression of CD44 variants expression. The results obtained for the CD44 and RHAMM protein expression were also found to be correlated with the obtained transcripts expression. However, the significance of these findings in tumourigenesis remains unclear. Findings from this investigation may help in the design and development of biocompatible implants with controlled surface properties to be used in cancer therapeutics; with medium and large HA polysaccharides being potential biopolymer candidates, useful for the development of novel therapies for highly invasive cancer. In addition, implications from this work can serve as a base for future research, and can lead to ideas for drugs and methods to be used in cancer therapeutic approaches.