Tribute to Dianne Rekow-Dental "Imagineer".

Elizabeth Dianne Rekow, BSME, MSME, MBA, DDS, MS, Certificate in Orthodontics, and PhD (1944-2022), was a dental science futurist pursuing brave new paths during her career. She was one of the pivotal scientists who initiated the CAD/CAM movement in the 1980s and went on to focus on digital dentistry for the rest of her career. Her professional contributions involved seven patents, 92 peer-reviewed publications, 10 book contributions, 31 proceeding contributions, and well over 100 national and international presentations. She was an avid supporter of women in science. Her greatest contribution was her expansive imagination. She served as 35th president of the American Association for Dental, Oral, and Craniofacial Research in 2006-2007 and 88th president of the International Association for Dental Research in 2011-2012. The present article reviews key elements of her career and includes testimonies from friends about her special relationships.

Effect of H2O2 Antiseptic on Dispersal of Cavitation-Induced Microdroplets.

The persisting outbreak of SARS-CoV-2 has posed an enormous threat to global health. The sustained human-to-human transmission of SARS-CoV-2 via respiratory droplets makes the medical procedures around the perioral area vulnerable to the spread of the disease. Such procedures include the ultrasonic dental cleaning method, which occurs within the oral cavity and involves cavitation-induced sprays, thus increasing the risk of pathogen transmission via advection. To understand the associated health and safety risks for patients and clinicians, it is critical to understand the flow pattern of the spray cloud around the operating region, the size and velocity distribution of the emitted droplets, and the extent of fluid dispersion until ultimate deposit on surfaces or escape through air vents. In this work, the droplet size and velocity distributions of the spray emerging from the tip of a free-standing common ultrasonic dental cleaning device were characterized via high-speed imaging. Deionized water and 1.5% and 3% aqueous hydrogen peroxide (H2O2) solutions were used as working fluids, with the H2O2-an established oxidizing agent-intended to curb the survival of virus released in aerosols generated from dental procedures. The measurements reveal that the presence of H2O2 in the working fluid increases the mean droplet size and ejection velocity. Detailed computational fluid dynamic simulations with multiphase flow models reveal benefits of adding small amounts of H2O2 in the feed stream of the ultrasonic cleaner; this practice causes larger droplets with shorter residence times inside the clinic before settling down or escaping through air vents. The results suggest optimal benefits (in terms of fluid spread) of adding 1.5% H2O2 in the feed stream during dental procedures involving ultrasonic tools. The present findings are not specific to the COVID-19 pandemic but should also apply to future outbreaks caused by airborne droplet transmission.

C5L2 Regulates DMP1 Expression during Odontoblastic Differentiation.

The presence of stem cells within the dental-pulp tissue as well as their differentiation into a new generation of functional odontoblast-like cells constitutes an important step of the dentin-pulp regeneration. Recent investigations demonstrated that the complement system activation participates in 2 critical steps of dentin-pulp regeneration: pulp progenitor's recruitment and pulp nerve sprouting. Surprisingly, its implication in odontoblastic differentiation has not been addressed yet. Since the complement receptor C5a receptor-like 2 (C5L2) is expressed by different stem cells, the aim of this study is to investigate if the dental pulp stem cells express C5L2 and if this receptor participates in odontoblastic differentiation. Immunohistochemistry performed on human third molar pulp sections showed a perivascular co-localization of the mesenchymal stem cell markers STRO1 and C5L2. In vitro immunofluorescent staining confirmed that hDPSCs express C5L2. Furthermore, we determined by real-time polymerase chain reaction that the expression of C5L2 is highly modulated in human dental pulp stem cells (hDPSCs) undergoing odontoblastic differentiation. Moreover, we showed that this odontogenesis-regulated expression of C5L2 is specifically potentiated by the proinflammatory cytokine TNFα. Using a C5L2-siRNA silencing strategy, we provide direct evidence that C5L2 constitutes a negative regulator of the dentinogenic marker DMP1 (dentin matrix protein 1) expression by hDPSCs. Our findings suggest a direct correlation between the odontoblastic differentiation and the level of C5L2 expression in hDPSCs and identify C5L2 as a negative regulator of DMP1 expression by hDPSCs during the odontoblastic differentiation and inflammation processes. This work is the first to demonstrate the involvement of C5L2 in the biological function of stem cells, provides an important knowledge in understanding odontoblastic differentiation of dental pulp stem cells, and may be useful in future dentin-pulp engineering strategies.

Topography Influences Adherent Cell Regulation of Osteoclastogenesis.

The importance of osteoclast-mediated bone resorption in the process of osseointegration has not been widely considered. In this study, cell culture was used to investigate the hypothesis that the function of implant-adherent bone marrow stromal cells (BMSCs) in osteoclastogenesis is influenced by surface topography. BMSCs isolated from femur and tibia of Sprague-Dawley rats were seeded onto 3 types of titanium surfaces (smooth, micro, and nano) and a control surface (tissue culture plastic) with or without osteogenic supplements. After 3 to 14 d, conditioned medium (CM) was collected. Subsequently, rat bone marrow-derived macrophages (BMMs) were cultured in media supplemented with soluble receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) as well as BMSC CM from each of the 4 surfaces. Gene expression levels of soluble RANKL, osteoprotegerin, tumor necrosis factor α, and M-CSF in cultured BMSCs at different time points were measured by real-time polymerase chain reaction. The number of differentiated osteoclastic cells was determined after tartrate-resistant acid phosphatase staining. Analysis of variance and t test were used for statistical analysis. The expression of prominent osteoclast-promoting factors tumor necrosis factor α and M-CSF was increased by BMSCs cultured on both micro- and nanoscale titanium topographies (P < 0.01). BMSC CM contained a heat-labile factor that increased BMMs osteoclastogenesis. CM from both micro- and nanoscale surface-adherent BMSCs increased the osteoclast number (P < 0.01). Difference in surface topography altered BMSC phenotype and influenced BMM osteoclastogenesis. Local signaling by implant-adherent cells at the implant-bone interface may indirectly control osteoclastogenesis and bone accrual around endosseous implants.

Influence of an LRP5 cytoplasmic SNP on Wnt signaling and osteoblastic differentiation.

The low density lipoprotein receptor-related protein 5 (LRP5) is a key determinant of bone mass, via the Wnt signaling pathway control of osteoblast function. This study examined human LRP5 signaling and the effects of an intracellular domain single nucleotide polymorphism (SNP: p.V1525A) on osteoblast differentiation and mineralization. Constitutively active LRP5 was constructed by deletion of the extracellular domain of LRP5 (LRP5DeltaN). Expression of LRP5DeltaN-V, which carries the allele p.1525V, induced higher beta-catenin/TCF-LEF activity compared to LRP5DeltaN-A, which carries the allele p.1525A. In a yeast two-hybrid assay, LRP5DeltaN-V also demonstrated a stronger interaction with AXIN than LRP5DeltaN-A. Expression of either of the alleles did not change cell proliferation. However, cells expressing LRP5DeltaN-V showed increased alkaline phosphatase activity and bone nodule formation compared to cells transfected with empty vector or LRP5DeltaN-A after osteogenic supplement (OS: beta-glycerophosphate and l-ascorbic acid) treatment. Cells expressing LRP5DeltaN-V revealed significantly increased bone sialoprotein (BSP) expression after 7 days of OS treatment and maintained elevated expression until day 21. Osteocalcin (OCN) mRNA levels were increased after 14-21 days of OS treatment in LRP5DeltaN-V expressing cells. LRP5DeltaN-V expressing cells demonstrated positive interaction with BMP-2 signaling of transcription at the SBE-luc promoter. LRP5 signaling is affected by the cytoplasmic SNP, p.V1525A. mRNA levels of Runx2 and Osterix were not affected by this SNP.

Anodic oxidation and hydrothermal treatment of commercially pure titanium surfaces increases expression of bone morphogenetic protein-2 in the adherent macrophage cell line J774A.1.

The surface property of commercially pure titanium (cpTi) was improved by forming a thin hydroxyapatite (HA) layer by anodic oxidation and hydrothermal treatment (HA/cpTi). We hypothesize that the adhesion of macrophages to HA/cpTi surfaces is important to the process of osseointegration. This study investigates the effect of adhesion of macrophages to HA/cpTi surfaces on the expression of bone morphogenetic protein-2 (BMP-2). The murine macrophage cell line J774A.1 was cultured on HA/cpTi and polished cpTi (S/cpTi). Macrophage cell adhesion was examined by SEM, 0-72 h following plating onto HA/cpTi and S/cpTi. BMP-2 gene expression was examined by RT-PCR analysis. The level of BMP-2 secreted into the supernatant was measured using an ELISA assay. The extent of macrophage adhesion increased with time on both the HA/cpTi and S/cpTi surfaces, with a" higher degree of spreading observed on HA/cpTi than onS/cpTi surfaces after 24 or 72 h. The ratio of BMP-2 mRNA was higher on HA/cpTi than on S/cpTi after 24 h (0.348 vs. 0, p < 0.05) and 72 h (0.584 vs. 0.189, p < 0.05). After 24 h, secretion of BMP-2 was detected in cultures grown on HA/cpTi, but not on S/cpTi. After 72 h, secretion of BMP-2 was detected in cultures grown on S/cpTi, but the levels were higher in cultures grown on HA/cpTi. These findings show that macrophages have the capacity to adhere to HA/cpTi endosseous implants and provide a source of osteoinductive cytokines that may play a key role in the process of osseointegration.

Effect of vitamin D pretreatment of human mesenchymal stem cells on ectopic bone formation.

Adult mesenchymal stem cells (MSCs) are used in contemporary strategies for tissue engineering. The MSC is able to form bone following implantation as undifferentiated cells adherent to hydroxyapatite (HA)/tricalcium phosphate (TCP) scaffolds. Previous investigators have demonstrated that human MSCs (hMSCs) can be differentiated to osteoblasts in vitro by the inclusion of vitamin D and ascorbic acid. The aim of this study was to compare the osteogenic potential of predifferentiated and undifferentiated bone marrow-derived, culture-expanded hMSCs adherent to synthetic HA/TCP (60%/40%) following subcutaneous engraftment in severe combined immunodeficiency (SCID) mice. During the final 3 days of culture, cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and antibiotics or media containing 25-mM calcium supplementation with vitamin D and ascorbic acid. Four weeks following implantation in SCID mice, scoring analysis of bone formation within the cubes revealed the absence of bone formation in unloaded cubes. Bone formation compared by a qualitative bone index was 7.23% for undifferentiated cells compared to 5.20% for differentiated cells. Minimal resorption was observed at this early time point. In this ectopic model, predifferentiation using a combination of vitamin D and ascorbic acid failed to increase subsequent bone formation by implanted cells. Following implantation of hMSCs adherent to an osteoconductive scaffold, host factors may contribute dominant osteoinductive signals or impose inhibitory signals to control the fate of the implanted cell. Predifferentiation strategies require confirmation in vivo.

Comparison of bone graft matrices for human mesenchymal stem cell-directed osteogenesis.

Scaffolds to support cell-based tissue engineering are critical determinants of clinical efforts to regenerate and repair the body. Bone tissue engineering requires materials that are biocompatible, well vascularized, mechanically suited for bone function, integrated with the host skeleton, and support osteoinduction of the implanted cells that form new bone. The aim of this study was to compare the osteogenic potential of bone marrow-derived, culture expanded human mesenchymal stem cells (hMSCs) adherent to different scaffolds composed of various calcium phosphates. Cells were loaded onto 2 x 2 mm cubes of coral calcium carbonate-derived apatite, bovine bone-derived apatite, synthetic hydroxyapatite (HA)/tricalcium phosphate (TCP) (60:40%), or synthetic HA/TCP (20:80%) and placed into the dorsum of SCID mice for 5 weeks. Subsequent histomorphometric analysis of bone formation within the cubes revealed the absence of bone formation within the coral-derived apatite and the bovine bone-derived apatite. Bone formation within synthetic HA/TCP scaffolds was measured to be 8.8% (+/-2.7%) and 13.8% (+/-3.6%) of the total tissue present for the 60:40% and 20:80% materials, respectively. Minimal resorption was observed at this early time point. Scanning electron microscopy evaluation of loaded scaffolds indicates that cell loading was not a variable affecting the different bone formation outcomes in these four scaffolds. In this ectopic model, different apatite-containing scaffolds of similar morphology and porosity demonstrated marked differences in their ability to support osteoinduction by implanted hMSCs. The necessary induction of hMSCs along the osteoblastic lineage may be dependent, in part, on the local microenvironment established by the scaffold chemistry and interactions with the host.

Titanium surface topography alters cell shape and modulates bone morphogenetic protein 2 expression in the J774A.1 macrophage cell line.

Macrophage cytokine expression significantly affects wound healing. Macrophage secretion of transforming growth factor beta 1 (TGFbeta1) and bone morphogenetic proteins (BMP) may affect osteogenesis at endosseous implant surfaces. The aim of this investigation was to determine the effect of commercially pure titanium (cpTi) substrate topography on adherent macrophage osteogenic and osteoinductive cytokine expression. J774A.1 murine macrophage cell adhesion was examined by scanning electron microscopy, 0-72 h following plating onto polished, machined, and grit-blasted cpTi surfaces. TGFbeta1 and BMP-2 gene expression by adherent macrophages was determined by the reverse transcription polymerase chain reaction. Macrophage adhesion increased with time on all surfaces and spreading increased with increasing surface roughness (polished < machined < grit-blasted). BMP-2 expression was not evident for cells adherent to polished cpTi at 24 h. In contrast, BMP-2 expression occurred at 24 h in cells adherent to machined and grit-blasted cpTi. BMP-2 expression was evident on all surfaces at 72 h and was greatest in grit-blasted titanium adherent cells. Increasing concentrations of cytochalasin B (0-50 microM) inhibited macrophage spreading and reduced BMP-2 mRNA expression, suggesting a relationship between cell shape and BMP-2 expression. This was further characterized using anti-beta1 and anti-beta3 integrin antibodies. The anti-beta1 integrin antibodies inhibited adherent macrophage BMP-2 mRNA expression. Anti-beta3 integrin antibody treatment only modestly reduced BMP-2 mRNA expression. Endosseous implant surface topography induced changes in macrophage shape that were associated with changes in BMP-2 expression in J774A.1 mouse macrophage cell line. This first demonstration of BMP-2 expression by cpTi adherent macrophages suggests that the macrophage may contribute surface-specific osteoinductive signals during bone formation at implanted alloplastic surfaces.

Macrophage cell lines produce osteoinductive signals that include bone morphogenetic protein-2.

Bone wound healing requires osteoinductive signals that are attributed to (the) bone morphogenetic proteins (BMPs). The cellular origin of such osteoinductive signals has only been partially elucidated. Because of the central role of the macrophage in cutaneous wound healing, we hypothesized that the macrophage could play a similar role in osseous healing. It was the aim of the present investigation to examine the possible expression of BMP by the macrophage, and to evaluate the contribution of macrophage products to an early step of bone formation modeled in an in vitro culture system. The synthesis of BMP-2 and BMP-6 by cultured human and murine macrophages was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). When human mesenchymal stem cells (hMSCs) were grown in conditioned media from J774A.1 cells, alkaline phosphatase expression increased. This induction was blocked by anti-BMP-2 antibody and by anti-transforming growth factor-beta1 (TGF-beta1) antibody. Modeling of the macrophage expression of osteoinductive signals by potential physiological situations was evaluated by treatments with lipopolysaccharide (LPS) or macrophage chemotactic peptide-1 (MCP-1). Macrophage BMP-2 expression was reduced by proinflammatory LPS stimulation (which was confirmed to induce release of the proinflammatory cytokine, TNF-alpha), and conditioned media from LPS-treated macrophages had no ability to increase alkaline phosphatase activity in hMSCs. This first study of macrophage BMP-2 expression indicates that the macrophage is capable of physiological regulation consistent with a key role in osteoinduction for osseous wound healing.

Incipient analysis of mesenchymal stem-cell-derived osteogenesis.

Tissue regeneration strategies invoke cell-based therapies for effective tissue formation. Current assessment of mesenchymal stem cell (MSC) directed bone regeneration during in vivo assays is dependent on histologic determination of bone formation. It was the aim of this study to determine the relationship between bone sialoprotein (BSP) expression and osteocalcin expression with subsequent osteogenesis occurring in MSC-based implants. RT-PCR assessment of human actin, collagen type I, BSP, and osteocalcin indicated that undifferentiated cells did not express BSP or osteocalcin. Three weeks following implantation, human BSP could be identified in RNAs isolated from the retrieved implants. For every implant from which human BSP cDNA was amplified, parallel implants harvested at 6 weeks demonstrated bone formation at the histologic level. This study confirms that, in the context of the severe combined immunodeficiency disease (SCID) mouse model, culture-expanded, cryopreserved human MSCs have osteogenic potential and demonstrates that implanted cell gene expression can reveal the early onset of bone formation.

Evaluation of a predictive model for implant surface topography effects on early osseointegration in the rat tibia model.

STATEMENT OF PROBLEM: Alterations in commercially pure titanium (cp Ti) implant surface topography can be made to increase bone formation or the interfacial shear strength of bone at the functioning implant. It is not known whether these 2 goals are congruent or mutually exclusive. PURPOSE: The aim of this study was to determine the effect of implant surface topography parameters of calculated biomechanical significance on the process of bone formation in a rat tibia model of osseointegration. MATERIAL AND METHODS: Implants (cp Ti grade IV) were machined and subsequently treated by grit blasting or grit blasting and 6.4 mol/L HCl. Measurements of surface roughness were made by atomic force microscopic analysis of similarly treated titanium disks. Cleaned and sterilized implants (12 machined, 12 with nonideal pit morphology, 12 with ideal pit morphology) were placed into the tibiae of 400-g male Wistar rats by using a series of drills, irrigation, and a self-tapping procedure. After 3 weeks, tibiae were harvested and processed and embedded in methyl methacrylate resin. Polished sections were examined by backscatter electron microscopy, and the percentage implant surface contacting bone was measured with the Scionics PC image analysis program. RESULTS. The implants possessing a proposed ideal pit morphology supported significantly greater bone formation at the implant surface (54% +/- 7% bone-to-implant contact [P<.003]) than the nonideal pit morphology (40% +/- 15%) or machined surfaces (34% +/- 6%). CONCLUSION: Implant surfaces with a proposed ideal pit morphology (which possess a calculated biomechanical significance) enhanced bone formation at early periods after placement in the rat tibia model.

Periapical abscess formation and resolution adjacent to dental implants: a clinical report.

The vitality of teeth adjacent to dental implants should be considered in the treatment planning of dental implants. Both the restorability of an endodontically treated tooth and the risk of infection of the adjacent implant are important factors in planning for success. Given the illustrated difficulties and difficulties associated with resolving periapical infections of teeth and implants, it is essential to define the vitality of teeth by careful pulp testing and to consider the integrity of existing questionable, endodontically treated teeth before implant treatment. The risk of periapical infection at teeth adjacent to implants must be minimized.

Variation in the spacer regions separating tRNA genes in Renibacterium salmoninarum distinguishes recent clinical isolates from the same location.

A means for distinguishing between clinical isolates of Renibacterium salmoninarum that is based on the PCR amplification of length polymorphisms in the tRNA intergenic spacer regions (tDNA-ILPs) was investigated. The method used primers specific to nucleotide sequences of R. salmoninarum tRNA genes and tRNA intergenic spacer regions that had been generated by using consensus tRNA gene primers. Twenty-one PCR products were sequenced from five isolates of R. salmoninarum from the United States, England, and Scotland, and four complete tRNA genes and spacer regions were identified. Sixteen specific PCR primers were designed and tested singly and in all possible pairwise combinations for their potential to discriminate between isolates from recent clinical outbreaks of bacterial kidney disease (BKD) in the United Kingdom. Fourteen of the isolates were cultured from kidney samples taken from fish displaying clinical signs of BKD on five farms, and some of the isolates came from the same farm and at the same time. The tDNA-ILP profiles separated 22 clinical isolates into nine groups and highlighted that some farms may have had more than one source of infection. The grouping of isolates improved on the discriminatory power of previously reported typing methods based on randomly amplified polymorphic DNA analysis and restriction fragment length profiles developed using insertion sequence IS994. Our method enabled us to make divisions between closely related clinical isolates of R. salmoninarum that have identical exact tandem repeat (ETR-A) loci, rRNA intergenic spacer sequences, and IS994 profiles.

A role for surface topography in creating and maintaining bone at titanium endosseous implants.

STATEMENT OF PROBLEM: A variety of claims are made regarding the effects of surface topography on implant osseointegration. Many in vivo and in vitro experimental observations have key limitations in their interpretations. PURPOSE: This review considers the major claims made concerning the effects of commercially pure (cp) titanium implant surface topography on osseointegration. Important findings of consensus are highlighted, and existing controversies are revealed. MATERIAL AND METHODS: This review considers many of the research publications listed in MEDLINE and presented in biomedical research publications and textbooks. RESULTS: Implant surface topography is not well defined in the marketplace or consistently reported among experimental studies. Many in vitro evaluations are not predictive of or correlated with in vivo outcomes. In some culture models, increased surface topography positively affects pro-osteogenic cellular activities. Animal models reveal modest increases in bone-to-implant contact and increases in the biomechanical interlock of the implant with bone for implants of increased surface topography. Existing information fails to define increased surface topography as a risk factor for peri-implant inflammation. CONCLUSION: Increased cp titanium implant surface topography improves the bone-to-implant contact and the mechanical properties of the enhanced interface. Growing clinical evidence for increased bone-to-implant contact at altered cp titanium implants confirms the temporally limited observations made in preclinical studies. In the absence of controlled comparative clinical trials, the aggregate experimental evidence supports the use of cp titanium implants with increased surface topography.

Renibacterium salmoninarum isolates from different sources possess two highly conserved copies of the rRNA operon .

The nucleotide sequences of the rRNA genes and the 5' flanking region were determined for R. salmoninarum ATCC 33209T from overlapping products generated by PCR amplification from the genomic DNA. Comparison of the sequences with rRNA genes from a variety of bacteria demonstrated the close relatedness between R. salmoninarum and the high G+C group of the actinobacteria, in particular, Arthrobacter species. A regulatory element within the 5' leader of the rRNA operon was identical to an element, CL2, described for mycobacteria. PCR, DNA sequence analysis, and DNA hybridisation were performed to examine variation between isolates from diverse sources which represented the four 16S-23S rRNA intergenic spacer sequevars previously described for R. salmoninarum. Two 23S-5S rRNA intergenic spacer sequevars of identical length were found. DNA hybridisation using probes complementary to 23S rDNA and 16S rDNA identified two rRNA operons which were identical or nearly identical amongst 40 isolates sourced from a variety of countries.

Estrogen-induced resistance to osteoblast apoptosis is associated with increased hsp27 expression.

Estrogen has been shown to protect osteoblastic cells from apoptosis. Similarly, estrogen treatment preceding heat shock elevates heat shock protein 27 (hsp27) expression and increases thermoresistance in the murine estrogen receptor-transformed SMER14 osteoblastic cell line. Forced expression of hsp27 expression in other cell lines limits apoptosis. The purpose of this study was to examine the effects of estrogen on staurosporine-induced apoptosis in the context of hsp27 expression. Cell viability was measured by the MTT assay. Early apoptotic events were examined by fluorescent microscopy by using FITC-conjugated Annexin V staining. TUNEL labeling was used to compare the number of apoptotic nuclei following staurosporine treatment of estrogen pretreated or untreated cells. Estrogen treatment increased SMER14 cell viability, but not ROS17/2.8 cell viability, in the presence of staurosporine. Estrogen treatment also reduced annexin V staining and DNA fragmentation. Similar treatment increased SMER14 cell hsp27 levels. The concurrent reduction in induced apoptosis suggests a possible estrogenic mechanism for increasing and/or maintaining the number of viable osteoblasts in bone.

Immunolocalization of stress proteins and extracellular matrix proteins in the rat tibia.

Stress proteins (heat shock proteins [hsps]) serve a number of protective functions, including protection from apoptosis and acting as chaperones during protein biosynthesis. For example, hsp 27 has been defined as a chaperone for the G3 domain of aggrecan, while hsp 47 is the chaperone for type I collagen. Separate cytoprotective roles for hsp 27 and hsp 70 have been demonstrated. The aim of this study was to define the expression of hsps in osteoblastic and chondrocytic cells of the growing rat long bone in relationship to the immunohistochemical localization of aggrecan, type I collagen and the presence of fragmented DNA that defines apoptotic events. Tibiae were harvested from Fisher 344 rats (n=6) and fixed in 10% buffered formalin. Samples were decalcified in 10% EDTA, bisected, and processed for histologic examination. Sections (5 mm) were immunohistochemically stained using a streptavidin-biotin detection method. Co-localization of hsps with apoptosis was achieved using the TUNEL procedure. In the rat tibia growth plate, aggrecan was generally distributed throughout cartilage and chondrocytes. However, hsp 27 expression was observed only in the lower hypertrophic chondrocytes. hsp27 was present in osteoblasts lining newly formed bone. hsp 47 staining was also prominent within these osteoblasts where collagen type I immunolocalization occurred. The inducible form of hsp 70 was localized to the osteoblastic cells lining new bone in the primary spongiosa. In cartilage, DNA fragmentation was restricted to the hypertrophic, hsp27-positive, chondrocytes. In contrast, DNA fragmentation was not co-localized with hsp27-positive osteoblastic cells of the primary spongiosa, although occasional apoptotic cells were identified. These results indicate that apoptosis is a mechanism by which hypertrophic chondrocytes are eliminated from cartilage prior to calcification, but that other mechanisms are also likely to be involved. They also suggest that hsps have cytoprotective and biosynthetic functions within osteoblasts and chondrocytes, but apoptotic signals may override these effects in some instances, resulting in apoptosis.

An N-terminal 33-amino-acid-deletion variant of hsp25 retains oligomerization and functional properties.

The mechanism(s) by which heat shock protein 25 (hsp25) protects cells from stress may involve one or more of the biochemical properties attributed to hsp25 and other small M(r) hsp. In this report, structural and functional properties of an N-terminal 33 amino acid deletion variant of hsp25 (termed hsp25.c) were considered by comparison with hsp25. 6-His tagged recombinant hsp25 and hsp25.c (termed (H6)hsp25.a and (H6)hsp25.c) were expressed and purified. Oligomeric proteins formed and possessed properties previously attributed to hsp25. The 33 amino acid deletion represented by hsp27.c did not affect the ability of the recombinant protein to act as an inhibitor of elastase, as a molecular chaperone in the refolding of denatured citrate synthase, or as an actin-binding protein. The overexpression of either hsp25 or hsp25.c, enhanced the stress resistance of stable transformed eukaryotic cells. This N-terminal variant protein may be used in further cellular and biochemical assessment of hsp25 oligomerization and function.