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David S. Howell, M.D.
BIOGRAPHY
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Introduction
David Sanders Howell was born October 4, 1923 in the northern New Jersey town of Montclair. His father worked in sales for Bradstreet Co. in New York City until his retirement, at which time the family moved to Tryon, North Carolina. David was then twelve and attended the nearby Asheville School for Boys preparatory school where, in addition to a conventional schedule, he studied oil painting. He was a pre-med student at the University of North Carolina, Chapel Hill from 1942 to 1943, with a minor in art history. An only child, David spent vacations beginning at age three in Boothbay Harbor, Maine in a home built on a cliff overlooking a bay leading out to the sea. Around age twelve, he spent the summer taking watercolor classes at the Anson Cross Art School in Boothbay (85B).
David graduated Phi Beta Kappa from Bowdoin College in 1944 with a B.A. in chemistry, and in 1947 he was awarded the M.D. degree from Harvard Medical School. He did an internship and residency at Brown Rhode Island Hospital from 1948-50 and married Margaret Blue of Coral Gables, Florida in 1948. He did a residency in pathology at the Hospital of the University of Pennsylvania from 1950-51. In 1951 he was accepted as a senior assistant surgeon at the National Heart Institute of the Public Health Service where, for the next four years, he studied and worked in biochemistry and physiology of the kidney and heart with methods for data measurement and statistical analysis of electrolytes, including sodium, potassium, serum proteins, magnesium, pH and bicarbonate.
Dr. Howell was named an Arthritis Trainee and Visiting Fellow in Medicine at Columbia Presbyterian Medical Center in 1954. Here he trained in the specialty of arthritis under Dr. Charles Ragan and was exposed to the teachings of the great Robert Loeb. Here he was taught and fully accepted the responsibilities of a career that included patient care, teaching, and research, to which he would add a career as an artist.
On July 1, 1955, Dr. Howell was appointed assistant professor of Medicine at the newly established University of Miami School of Medicine and Director of the Arthritis Division at Jackson Memorial Hospital. The Arthritis Division flourished under his leadership and with the help of the full-time faculty he selected, including Drs. Harvey E. Brown, Jr., Roy D. Altman, and Norman Gottlieb. An intense program of teaching activities was developed which saw more than 70 medical residents, who in two or more years of specialty training, committed their lives to rheumatology. Some of these and other well trained rheumatologists then faithfully served as voluntary faculty at the bedside, along with full-time faculty, training students, housestaff and residents. Beginning in 1957, two Arthritis Clinics at the Veterans Administration Hospital and three at Jackson Memorial Hospital were serviced weekly by the Arthritis Team. Thousands of indigent and private care patients from all over the world were seen and referred annually to the Arthritis Team for more than 40 years, a mark of their own distinguished careers. In 1967, Dr. Howell was promoted to Professor of Medicine, and in 1978 he was appointed Professor of Orthopedics.
In contrast to the teaching and patient care activities which were handled adroitly by the Arthritis Team, Dr. Howell focused on research as he built the academic arthritis research program at the University of Miami. His research activities, which had begun in 1951 at the National Heart Institute, continued with a 1958-1962 Markle Foundation Scholarship and a 1960-62 Fellowship at the Biochemistry and Cell Research Institute of the Nobel Institute in Stockholm. Starting with a training grant in 1958 and ending with a 10 year MERIT award, support from the NIH continued uninterrupted for 42 years and included six consecutive five-year NIH grant renewals. Dr. Howell also received four consecutive five-year VA investigatorship awards plus four extra years. All grant and renewal requests, except one, were written by Dr. Howell and funded without revision.
Dr. Howell’s main interest of research was osteoarthritis and his contributions to this field form an important part of this autobiography, but, with the exception of his personal research conducted between 1957 and 1965, all studies were team efforts that combined the work of mature scientists, trainees and collaborators. Dr. Howell’s specific interests were 1) electrolytes and calcification in the growth plate which often reopens in osteoarthritis, with a particular emphasis on model mechanisms of mineralization in growth plate cartilage septae as studied by micropuncture techniques; 2) pyrophosphate generation by chondrocytes and elevated levels of NTP pyrophosphorylase in chondrocalcinosis and osteoarthritic human subjects; and 3) proteoglycan aggregation in relation to articular and growth cartilage physiology.
Throughout the following text, references which include Dr. Howell’s involvement appear in parentheses and link to the book, journal, or abstract section of his bibliography elsewhere on this website. All other references appear in brackets and link to the bibliography which appears at the end of the text. This account of his work provides only a bare minimum of such references, and literature reviews of Boyan, Boskey, Howell and others are recommended. In addition to work published in symposia and books, journal articles involving his electrolyte growth plate physiology career include references 23, 24, 30, 32, 33, 36, 37, 40, 41, 42, 47, 48, 49, 50, 54, 55, 56, 60, 62, 64, 67, 71, 73, 75, 79, 81, 86, 87, 95, 103, 105, 106, 124, 146, 148, 150.
Early Proteoglycans and Methods Assembly, 1960-1968
A five year Markle grant supported Dr. Howell in the study of calcification physiology and related proteoglycans as the basis of a long program in osteoarthritis. The study began with a profile of electrolytes in the cartilaginous plate of growing ribs, the first profile of all major electrolytes studied by cross-sectional zonal analysis of a cartilaginous growth center, i.e. bovine costal cartilage. The measurement of multiple parameters related to mineralization showed a high positive correlation of sodium content with proteoglycan sulfate levels. This indicated that the major cation for proteoglycans is sodium, although some extracellular potassium is likely in the late hypertrophic zone where there was some evidence for storage of calcium (23). These findings were confirmed, in general, in the porcine growth plate studies of Roy Wuthier published in 1971 [1] and the electron probe work of Hargest, et al published in 1985 [2]. These first morphological analyses showed an enormous increase in hypertrophic cell volume between the proliferating and hypertrophic cell zones (23).
In another early study, evidence for mineral deposition on polyvinyl sponges placed under the skin in the rat (24) was presented as a deterrent to usage in plastic surgery. “Concepts of calcification,” published in 1963, reviewed theories on the regulation of biological mineralization (29). These early examples of research writings translated to patient care, and synthesized for educational purposes set a tone which was continued for the next four decades.
Some Markle studies were carried out at the Cell Research Institute of the Nobel Institute in Stockholm because of its unique facility for x-ray elemental analysis, a method prior to the electron probe for measuring sulfur and phosphorus, as well as total mass, in 3 x 50um x 20um sites in a tissue at the light microscopic level. This x-ray method of Lindstrom and Engstrom [3-4] was used to map sulfated proteoglycans and mineral in cartilage to study calcification of upper tibial growth plate and costal growth septa. By using collimated monochromatic x-rays and knowledge of elemental absorption coefficients, elemental concentration in a septal histological slice could be calculated from densitometric measurements on Kodak film. Bostrom showed that over a period of incubation 35 sulfate incorporated >95% of the label into sulfated mucopolysaccharides of cartilage organ cultures [5].
Staining by Sudan Black of calcifying regions for phospholipids [6-7] and x-ray analysis for sulfur (32) demonstrated that phospholipids and sulfated proteoglycans respectively are elevated in calcifying sites, such as the border of dentinal mineralization in calf teeth and the border of osteoid and long bone periosteal new bone formation and calcifying cartilage septa, as compared to adjacent non-calcifying sites (32). These findings of increased levels of sulfur in mineralizing bone margins were confirmed by microscopic analyses by Baylink, et al [8]. Finding lysophospholipid within calcifying sites demonstrated the degradation of phospholipids at these sites, which was considered evidence of matrix vesicle membrane breakdown (36) by endogenous phospholipase A2.
Data from a study on the mineralization rate (39) were subsequently reviewed (37B). Over a nine hour period the new mineral content of septa attained the level of 37% wet weight, assuming a hypertrophic cell, and its adjacent septum entered the calcified zone every three hours. In line with other papers at the time on papain effects on growth plate in vivo [9], papain treated rachitic rat growth plates showed a several fold reduction of sulfur as an index of proteoglycan sulfate removal. Mineralization was not reduced on healing rickets in the papain treated growth plate sites. These findings showed severe reduction of sulfated proteoglycans, which did not seem essential for changing normal mineralization (37). In retrospect, these findings resemble histological results in genetically deficient animals with disturbed proteoglycan sulfation, except, being short term, they did not show consequences on structural biomechanics.
The early Markle studies showed a rapid rate of mineral growth with localization to histological sites enriched in phospholipids and sulfated proteoglycans, along with a loss of proteins in growth plates. In collaboration, Karl Meyer in New York found in rachitic calf cartilage septa chondroitin 4-sulfate and a trace of keratan sulfate (33). These were among the earliest measurements that most of the sulfur in cartilage was as glycosaminoglycan sulfates.
Ultracentrifugation and chlorophosphanazo dye were used for a new miniaturized method for micropuncture fluids to determine free versus bound calcium. J.C. Pita, an associate in Miami, invented a microcuvette and optical bench which allowed for the measurement at ultramicro levels of sample calcium, free in solution and bound to protein, using an ultracentrifugation technique (38). The next two sections describe the important findings made possible by the new methods assembled in Stockholm and Miami.
Micropuncture I: Fluid in Growth Plates, Nasal and Articular Cartilage, 1968-1989
Methods worked on at the National Heart Institute and in Stockholm and Miami led to the discovery of a fluid phase and its electrolyte characterization, compared to whole tissue, at the calcifying front of rat tibial growth plates (40). Howell created the experimental designs and Julio Pita adapted and miniaturized macroscale methods for the first time to permit analysis of electrolytes in 1-20 nl samples. Howell had attempted micropuncture in Stockholm, but failed due to ballistocardiographic fracture of pipettes. Pita also developed adaptations of ultracentrifugation, as well as UV and fluorescence photon counter methods. Twenty two new methods were developed or adapted for the micropuncture fluid analysis of calcifying sites in rat growth plates and in lapine growth and nasal cartilage (22B). Several methods were published in Analytical Biochemistry or Analytical Chemistry.
The first paper measured electrolytes using the new methods in Miami and helium glow photometry at NIH in 20 nl samples of fluid aspirated in vivo from or adjacent to a septal calcifying site in rats, on a diet normal in calcium, low in phosphorus, and absent vitamin D and in healing rickets. This permitted data collection before and after the appearance of mineral (40). The measurement of bound and free calcium and phosphate provided the first evidence that there is not a generalized pump mechanism in extracellular open space to raise cartilage fluid level of “free” calcium and phosphate to cause precipitation of mineral above plasma levels. There was elevation of phosphate in the micropuncture fluid of rachitic rats compared to concurrent plasma samples in rickets. There was a three-fold elevation of nucleotides in all micropuncture fluids, a potential source of free phosphate. A three-four fold elevation of alkaline phosphatase and an eight-fold elevation of ATPase were later shown (81), as was an accumulation of phosphate in incubated microscopic samples. This finding provided the first micropuncture direct support that hydrolytic enzymes released phosphate and pyrophosphate from nucleotides at sites in the presence of presumed calcium storage. Micropuncture levels of pyrophosphate in the 10-6 molar range were found in long bone culture to stimulate mineral deposition by Anderson [23].
In one paper, studies were made of fluid from calcifying sites for inhibitor properties and screening tests wee done for nucleating factors and macromolecular inhibitors of mineralization (42). A subsequent paper showed evidence for the role of the R-2 proteinpolysaccharides of Schubert in the regulation of mineral phase separation in calcifying cartilage (47). Next, a study was designed to use a carbonic anhydrase inhibitor acetazolamide in vivo in rats to reduce the alkaline pH of fluid at calcifying sites. This study provided evidence of a fast flow system of extracellular fluid along the cells that provided a probable source of calcium for either storage and release in vesicles, or release from early apoptotic cells at sites of mineralization. Such a flow remains unproven, but was suggested by identical levels of partial pressures of carbon dioxide from the calcifying septa versus the level in adjacent capillaries measured on 3-5 nanoliter samples (49).
The carbonic anhydrase method in this study (49) was not sensitive enough to detect hypertrophic cellular enzyme, in contrast to methods later used by Carol V. Gay at Penn State. Gay and associates utilized both an immunologic method and triated acetazolamide to localize carbonic anhydrase in hypertrophic cells (95). The collaborations with Gay and colleagues were instigated by the elevated pH of growth plate cartilage fluid. The importance of the alkaline pH in cartilage fluid is that less mineral could be deposited in these sites without removal of hydrogen ion in large amounts, complicated by lowering of pH by weight bearing pressure [11]. Howell believes this excess hydrogen ion is carried out either by storage in the chondrocytes, until the cells are finally destroyed by apoptosis and the acid released into the circulation, or it is carried by a lymph flow system and circulated back to the metaphysis. Maren found acetazolamide caused disturbances in electrolyte metabolism followed by compensatory responses [10]. Some of these would probably also modify pH in the acidic direction but bone formation was not mentioned.
In 1973 Cuervo, Pita and Howell showed a precise small amount of calcium binding (55) that was in keeping with the classical studies of Farber and Schubert of chrondroitin sulfate in synthetic lymph. Howell believed that cartilage proteoglycan free in solution does not sufficiently bind calcium for the important storage mechanism shown in this paper, because of lymph sodium at physiological levels. Proteoglycans are free in solution as sampled in micropuncture fluid. According to Farber and Schubert, calcium binding to chondroitin sulfate in human lymph is determined by the ratio of sodium to calcium and their binding constants. Here the proteoglycan acted the same as chondroitin sulfate in free solution (55).
Bowness had postulated that a heavy fraction of proteinpolysaccharide bound to fibers in the growth plate hypertrophic zone pericellular matrix did bind calcium [12]. In later in vitro studies of proteoglycans, Hunter showed ion exchange under favorable conditions of flow and high metastable lymph calcium x phosphate generate calcium phosphate mineral [13]. Neither demineralized whole tissue nor pooled small samples of fluid aspirated from already calcified cartilage zones in rat and rabbit growth plate showed any vestige of >100S aggregates or r2 fraction (47). The studies of Dietwiekowski, Campo, Lohmander, Buckwalter and Rosenberg, Erlich, Boskey, Mankin, and including Cuervo, Howell and Pita (55) favored proteoglycan aggregates to inhibit calcification, but probably more than one function is likely.
The 1970 paper (47) was also the first in a line of studies from Howell’s lab in which evidence was found for a superaggregate proteoglycan inhibitor of early mineral expansion. Pita and Muller’s collagenase associative extraction method demonstrated that the matrix gla protein inhibitor, later discovered by others, is not the only bona fide major inhibition factor. Inhibitory aggregates are abundantly present in the whole tissue and fluid phase before the calcifying front, and absent in the calcified zone. In 1974, together with K. Kuettner and others, predominant proteoglycan was shown, in the region at the calcifying front where micropuncture fluids are collected, to be a superaggregate in the ultracentrifuge, >100S, over double the value of a 50S aggregate (61). This was later confirmed by Buckwalter, Pita, Muller and Nessler as over a purified double sized molecule [14] (not only by ultracentrifugation but by electron microscopic monolayer study), and predictably larger molecules were made by Tang, Buckwalter and Rosenberg [15], depending on link protein concentration. Thereby, an over double sized molecule of aggregate was found in native tissues by collagenase extraction. Great efforts were made to exclude artifacts in the collagenase extraction method and none were found. Based on data of Muller, et al [16], this >100S aggregate is found enriched in midzonal articular cartilage in extracellular sites.
Muller, et al postulated a relationship of this over double molecule to articular cartilage protection against compressive force injury and its singular early loss during osteoarthritis development in the ACLT canine model, when smaller aggregate profiles classically extracted showed no changes. This shed light on its likely role in the rat and rabbit growth plates, where Dean, et al (110), Poole’s group in rats [17], and Brown et al in rabbits [18], showed abundant collagenolysis at or before the calcifying front. It seems likely in view of the discussion of Tang, et al [15] in respective joint cartilage that heavy collagenolysis and minor proteoglycanolysis in the –Pi,-vita D rachitic growth plate may account for the abundance of superaggregates found at the sites of mechanical maximal vulnerability as studied in rats.
Based on these studies, that the collagenolysis was sufficient to create weakened tissue, it could be postulated that in response local cells synthesize the over double aggregates to be inserted into the spaces and act as physical buffers of the weakened collagen network. Such superaggregates might play an important biomechanical role in distal growth plates at sites in septa deprived of some structural integrity by collagenolysis. In 2006 it appears still to be a possible explanation for the presence and location of superaggregates.
Howell also postulated that the >100S aggregates were active, at least when free in solution in vitro, to inhibit mineralization, along with the generally accepted matrix gla protein, which is backed by strong in vivo evidence. Superaggregates studied in rickets for its enlarged hypertrophic cell zone are quite resistant to proteolysis, but are rapidly disaggregated by endogenous lysozyme or added lysozyme, and are possibly related to the high alkaline pH. The role of lysozyme is, therefore, still a possible alternative to explain the rapid removal of superaggregates at the calcifying front of growth plate cartilage in rats and rabbits and the restoration of aggregation with chitotriose (66). In healing rickets in vitamin D deficiency, Hertquist showed [9] there was retarded synthesis and retarded degradation of proteoglycans, the same as found by Dean, et al in 2003 with regard to their enzymic breakdown in healing rickets (174).
Micropuncture II: Predentine, Dentine EHDP Rickets, MV Protease Hepatocyte Growth Factors, 1976-1994
Healing of rickets with intraperitoneal sodium phosphate caused a sharp peak of phosphate in the micropuncture fluid and, at the same time, mineral deposition on electron microscopy. Howell believed that calcium x phosphate ratio reached levels of high metastability, but not levels sufficient to cause mineral precipitation per se (56, 67). Small araldite chambers devised by Ralph Alvarez were used in this study (67) to deposit and centrifuge micropuncture fluid samples. The sediments were sealed off and the araldite sectioned for electron microscopy. These samples, studied on transmission electron microscopy by Juana Alvarez, showed trilaminar membraned particles consistent with matrix vesicles, and also particles of collagen in small amounts.
With further experience Howell believed that the collagen, matrix vesicles and later sediments proved a biopsy-type component accompanying the micropuncture fluid aspiration. The fluid volume per se of 10 to 20nl could represent sampling around cells, but the tiny amounts of sediment probably were localized to the septum pierced by the sharpened micropipette. Photographs of troughs in the septa shown by proteoglycan staining were also consistent with a local biopsy of sediment by Croxen and Kuettner.
Due to tiny sampling and high complexity of samples by micropuncture study, the distinction between formation of new mineral, i.e. binding between calcium and phosphate, is unrelated to binding in these studies. Howell brought in a chemist and biologist, Luciano Blanco, to develop an ultramicrodialysis alternative method for measuring calcium. The technique Blanco devised used lanthenum as a critical reagent for reproducible results which could be obtained from less than 5 nanoliter samples (80).
Using both ultracentrifugation and the dialysis method, calcium binding was shown to a fraction in the top layer of micropuncture fluid following ultracentrifugation to get rid of inhibitory proteoglycans. This fraction was capable of nucleating mineral within 3-5 hours, according to principles developed in that era. The factor resisted acidification to pH 5.8, and was not composed of apatite crystal (81). The factor was soluble in chloroform-methanol, resistant to phospholipase D, degraded by phospholipase C, and had a composition similar to matrix vesicles in respect to content of phospholipids and high phosphatidylserine. It was extremely important that Cieslak, Boyan and Howell found in these fluids a microphospholipid profile (16A), typical of matrix vesicles described by Boyan and Boskey [19] as a membrane fraction with strong nucleating properties labeled CPLX.
The first micropuncture and microanalysis of predentine fluid also showed no general elevation of calcium or phosphorus sufficient to cause mineral precipitation in calcifying site fluid (125). This study was led by P.A Larsson, professor of dental pathology at the University of Lund and chief dental pathologist in Sweden, during a six-month sabbatical in Miami, and assisted by Claude Le Chane, the chief of kidney research at Harvard Medical School. This study was also a reinvestigation of electrolytes in 1 nanoliter samples of micropuncture cartilage fluid studied by electron probe electrolyte analysis, instead of the helium glow photometry. The levels of calcium, phosphorus, sodium, and chloride were within a range a little lower, not higher as hoped for, than those found by Howell’s previous micropuncture studies. Samples as small as 300 picoliters were studied, which had to be within about four to eight cells of where calcification was occurring. It is assumed that proteoglycans in the probe samples and/or the high protein level there caused some type of interference in data collection. However, the results of the reinvestigation were sufficient to support prior data.
Proteases in the Rat Growth Plate, 1985-2002
The first demonstration of biochemically defined collagenase in the rat growth plate (110) stemmed from the obvious likelihood that since chondrocytes enlarged 7 to 10 fold over a period of several hours in the growth plate, the extracellular matrix accompanying the cellular enlargement must remodel, and collagenase would have to be secreted and activated by the chondocytes to degrade the matrix around the enlarging cells. The level of collagenase in an enormous upregulation without comparable upregulation of TIMP (135), measured by methods of Woessner and Dean, was found comparable to that of the involuting rat uterus. A.R. Poole and collaborators over the next decade developed key immunological methods to quantify and localize collagenase-produced collagen breakdown.
This first biochemical demonstration of collagenase in the growth plate (110) was followed by the first suggestion of immunoreactive collagenase in the rat growth plate (130) and by reactivity of rabbit growth plates to an anticartilage collagenase [18] where Hunziker had demonstrated cells were enlarging and elongating to accelerate growth [20]. Evidence of proteoglycanases working at least to some extent in the rat normal growth plate is indicated by Axelsson’s finding of a faster half-life of proteoglycans than the half-life of growth plate loss and replacement (136). Such losses could be septal proteoglycan associated with mineralization. Loosening of the hypertrophic zone network and presence of aggrecanase were found by Plaas and Sandy [24].
Another study showed, for the first time, the presence of the gene for hepatocyte growth factor and hypertrophic cells of the distal growth plate of normal rats and rachitic rats. There was also upregulation of the C-met receptors. Treatment of hypertrophic chondrocytes in culture with rat hepatocyte growth factor increased alkaline phosphatase activity several fold. Hepatocyte growth factor protein was identified (162), and a later study by J. Martel-Pelletier showed that truncated isozymes were made in human equivalent tissues, but normal genes found in adjacent osteoblasts might cause formation and diffusion of hepatic growth factor into cartilage [26].
Whereas the brilliant work of the Boyan, Schwartz, Dean and Silva group in San Antonio showed a marked response of the resting cell zone to 24,25(OH)D3, and of the growth zone to 1,25(OH)2D3 in rat rib cartilage, others found 24,25(OH)D3, a more potent stimulator of mineralization in the growth zone than 1,25(OH)D3. Faster healing with 24,25(OH)D3 in both bone and cartilage was shown in a study in rachitic tibial growth plates and metaphyses by Atkin, et al than with 1,25(OH)2D3 (146).
Studies of rat growth plate and metaphyseal bone cells showed that matrix vesicles produced by osteoblasts, as well as by chondrocytes, contained MMP2 and MMP3 in rats (148, 154). These enzymes breakdown inhibitors of local calcification or make space for new mineral at calcifying sites (154).
Together with Dean, Boyan and others the first refereed paper to show important increased levels of IL-1ß in growth plate cartilage, especially in the hypertrophic cell zone, was published (170). For this study, the action of an assay of IL-1 was assessed by its stimulation of the degradation of proteoglycan in articular cartilage by extracts from growth plate. Antibodies were also used to measure or block the action of IL-1ß. In the last study with Dean, Boyan and others, collagenase and proteoglycanase activity, which was high in the hypertrophic zone of untreated rachitic rats, showed a significant reduction of both types of activity on treatment with vitamin D. The predominant action of 1, 25 dihydroxyvitamin D3 was on collagenase, and of 24, 25 on the proteoglycanase effect (174). A reduction of metalloproteinase activity over a physiologic range of dosage of vitamin D metabolites was also confirmed in cultured chondrocytes of growth and resting zone. The final publication on calcification, with an emphasis on genetic studies, was a review published in 2002 (178).
Pyrophosphatate, Pyrophosphatase, NTP Pyrophosphohydrolase, 1972-1984
The first analysis of normal pyrophosphate levels in human synovial fluid (58) and in micropuncture fluid was performed using a pyrophosphate method developed by Muniz (12B). High levels of pyrophosphate in synovial fluid from osteoarthritis cartilage were then demonstrated using the Muniz method and explained by the secretion of chondrocytes (64, 75). High levels of alkaline phosphatase were found in aging osteoarthritic cartilage only at the tidemark zone, but the alkaline phosphatase was exceptionally low in osteoarthritic lesional cartilage (70).
A definitive paper showed pyrophosphate generated from nucleotides in human articular cartilage and documented its significant increase in osteoarthritis and calcium pyrophosphate deposition disease (87). Various nucleotides were shown to produce pyrophosphate (106) via elevated levels of an enzyme. Evidence was found that NTP pyrophosphohydrolase is an ecto-enzyme (105), concurrently with Ryan [22]. An ecto-enzyme was postulated from the hydrolyzing enzymes that were released around articular cartilage to cause the production of pyrophosphate, probably in the growth plate, and from the effect of trypsin produced on chondrocyte subcellular fractions analyzed for NTP pyrophosphohydrolase.
It was postulated that the failure of hydrolysis of pyrophosphate in primate articular cartilages, in comparison to the growth plate where there was rapid hydrolysis, was due to low alkaline phosphotase levels in mid-zonal articular cartilage. Thus, a theory was advanced on how NTP pyrophosphohydrolase on the surface of chondrocytes could generate pyrophosphate from extruded nucleotides, probably caused by injury of the faulty cartilage matrix. This would lead not only to release of pyrophosphate, but also 5’AMP. A high level of 5’AMPase was found and postulated to accelerate pyrophosphate formation by removing the other end product, 5’AMP.
A current view that alkaline phosphatase and NTP pyrophosphohydrolase together control the rate of normal bone mineral deposition is derived from studies of mice with deletion of one or both genes led by HC Anderson [23]. Clearly, the importance of this enzyme more than two decades later transcended all expectations. The current theories of how pyrophosphate mineral is deposited in human cartilage rest on this theory. The independent work of C.F. Bonting and R.G. Russell, Ryan, Pritzker and others, also contributed matrix vesicle functions of NTP pyrophosphohydrolase in osteoarthritic cartilage and Hsu in growth plates [21].
Proteoglycan Aggregation in Normal and Osteoarthritis Cartilage, 1979-1996
Howell’s first osteoarthritis profile of proteoglycans used the Moskowitz model of osteoarthritis with medical meniscus alteration. The profile demonstrated loss of proteoglycan aggregates at a level of high significance and correlated with histologic changes (83). A study in normal rabbit articular cartilage used rate-zonal centrifugation, a modified ultracentrifugation method developed by Pita, that is superior to the velocity gradient method. This method documented the presence of 10 or 15% of superaggregates (90). This method was also used to more clearly separate and then compare proteoglycan monomers, nonlink stabilized aggregates, and link protein stabilized aggregates in normal versus osteoarthritic cartilage using the Moskowitz model (108).
Manicourt, a funded investigator who joined Howell’s team to earn a PhD in the proteoglycans field, compared Pita’s purified bacterial collagenase associative method of isolating proteoglycan aggregates instead of the Sajdera Hascall (SH) method of hypertonic extraction of proteoglycan aggregates. The Pita method clearly showed a superaggregate component of 15 to 20% of the total proteoglycan, more than 100S of normal dog femoral articular knee cartilage. The dissociative extraction method showed the free monomers and small to average size aggregates, but no sign of a superaggregate (115). The Pita method also extracted more protein and found no link protein bound to aggregates in the surface zone (126). Two and a half times more link protein was present in the large than in the small aggregates, and twice as many chondroitin sulfate molecules, shown by Buckwalter, et al [16], were in the large than in the small aggregates in preparative samples.
The collaborations with Pita demonstrated two classes of proteoglycan aggregates, a large and a smaller sized aggregate, in the micropuncture fluid. In the rat growth plate there was a single link protein demonstrated that could be dissociated from the aggregates found. The large, but not the small, proteoglycan aggregates were shown to bind the calcium phosphate mineral and retain mineral at low centrifugation speeds from micropuncture fluid, in a manner similar to the R-2 fraction of Maxwell Schubert. Characterization of the large and small proteoglycan aggregates has continued up to the present time.
Strong evidence that these aggregates were not an artifact was their separation, without any preparative procedures whatsoever from micropuncture fluid, the duplication of this separation and same size distribution s value profile by Pita’s associative extraction method and, after collagenase treatment, from the same growth cartilages. The fact that in cartilage atrophy in the dog the small aggregates are reduced, instead of the large aggregates, again indicates a major metabolic difference between the two disorders.
The loss of superaggregates was found to occur early in experimental osteoarthritis in dogs and more severely from high than low weight bearing sites (134). The loss of the large aggregates routinely in osteoarthritic cartilages in ACLT, as well as Moskowitz osteoarthritic rabbit articular cartilages, make these extremely interesting profiles for structural proteoglycan analysis as shown in 2005 by Manicourt’s group [29]. Superaggregates were found to be largest and most abundant in high as opposed to low weight bearing sites in canine knees (142). Changes in size distribution profiles of proteoglycan aggregates in normal versus the ACLT dog model knee cartilage, as well as the sling disuse model, were reviewed. The sling model permitted study of the earliest and hopefully most fundamental changes in disuse immobilization (149). The quantitative polymerase chain reaction assay was used to study aggregan and link protein gene expression in cartilage (161) in a study of Dr. Ratcliffe. In studies of growth plate, proteoglycan content is reduced greatly as the calcified zone is approached and link protein is low [25] with aggrecanase demonstrated [24].
Mechanical Properties and Biochemical Changes in Proteoglycans in Osteoarthritis and Disuse Atrophy, 1981-2002
Increased swelling and reduced superaggregate size in the midzone in experimentally induced osteoarthritis in ACLT canine cartilage were detected by surface biomechanical studies and by the Maroudas method of equilibrating normal saline with articular cartilage slices to detect degree of damage to the collagen network from swelling (104).
The Mow isometric tensile apparatus was used to study kinetic swelling properties in normal, unfibrillated and fibrillated samples of human osteoarthritic knee joint cartilage. Measuring the peak stress, stress relaxation and sodium diffusion constant in these samples showed that height of peak stress correlated strongly with proteoglycan content and stress relaxation with collagen content in normal cartilage, but much less so in fibrillated cartilage. These parameters were related to intrinsic general mechanochemical material coefficients that describe the swelling behavior of cartilage (117). The Mow apparatus was also used to study equilibrium swelling and to correlate it with high and low weight bearing sites and grades of osteoarthritis (123).
Mow’s biphasic theory was illustrated in a data analysis of reviewed papers. Along with early biomechanical changes, the loss of large aggregates was found to appear early and mostly in high stress medial femoral condyles in the anterior cruciate ligament transections (ACLT) model (152). Proteoglycans were reduced in both animal models. Slow sedimenting aggregates were reduced in the disuse model, and slow and fast sedimenting aggregates were reduced in the ACLT instability model. Hyaluronan, reported reduced in human hip osteoarthritis, was always reduced in the instability model, perhaps accounting for the irreversibility of the osteoarthritis model (156).
A study of equilibrium, transient, and dynamic shear behavior of articular cartilage in the ACLT model showed a fall of all cartilage moduli tested in dynamic and equilibrium shear and compression, beginning six weeks after surgery (157). A torsional shear study in the ACLT model confirmed the large reductions of moduli found in the biphasic indentation study and showed increased water content, with changes in flow independent elastic behavior (160). Increased water and stiffening of cartilage were found in the patellar groove following periods of joint disuse and disuse with remobilization (167).
Surface and deep zones in the patellar study showed only minor changes. Surface cartilage showed degenerative changes around cells, and considerable deep level strains and stresses developed in a histometric study of very mild disuse atrophy in the sling model (175). The histometric method developed by Setton was also used to analyze multiple mechanical parameters in deep cartilage layers (179). The V.C.Mow mechanics analysis of cartilage structure, as well as the studies of his protégé L. Setton and his research fellows and pre-doctoral students, made history on early and mildly severe osteoarthritis and disuse immobilization.
Cartilage Proteases in Normal vs. Osteoarthritis Cartilage, 1973-1984
Studies conducted with Roy Altman on loss of the R2 fraction of Schubert in human osteoarthritic cartilage (53), which Howell still believe is an index of loss of fast sedimenting aggregates, led to an interest in the possible role of proteases in cartilage degradation. To pursue this new interest, Dr. Sapolsky obtained his PhD with Dr. Fred Woessner, a leading international authority on proteolytic enzymes based at the University of Miami. A first report by Sapolsky in this collaboration was the characterization of the action of cathepsin D in human articular cartilage in autopsied patellas (52). Howell’s lab team’s earlier miniaturized methodologies, histology and cartilage physiology expertise and techniques, and osteoarthritis tissue sources, helped Sapolsky and Woessner to generate the first data on extraction and characterization of an enzyme active at neutral pH that degraded proteoglycans.
The characterization of the action of cathepsin D was shown to be confined to chondrocytes and followed by other studies on autopsied patellas that detected and characterized neutral and acid metalloproteinases. In another 1976 publication, neutral metalloprotease was purified 900-fold and showed evidence of multiple isoenzyme forms, and proteoglycans were shown to be optimally degraded at pH 7.25, with total inhibition by a2 macroglobulin and by 1-10 phenanthroline (74). The collaboration work on degradative enzymes in osteoarthritic human articular cartilage was featured in the 1975 Pemberton Lecture (63).
A review on the pathogenesis of osteoarthritis showed that the neutral protease of interest was insensitive to inhibitors of serine or cysteine proteinases and partly inhibited by ethylenediaminetetraacetic acid (69). Another review featured diagrams of alternative pathophysiological pathways of osteoarthritis (72). Woessner reviewed the complex analytic problem of different proteases, that required an assortment of substrates and different chromatography schemes before the advent of current gene and immune-based approaches (77). Sapolsky, still under Dr. Woessner in his postdoctoral stage, later purified 1200 gm of human articular cartilage 1400 to 2400 fold by diethylaminoethyl and carboxymethylsephadex chromatography. He resolved the protease into four 4 bands, 24 to 27 thousand daltons, which degraded aggregan to 3s fragments by disc gel electrophoresis and isoelectris focusing (96).
Woessner’s work in enzyme chemistry led to identification of human articular cartilage stromelysin in 1989 [27] and TIMP there in 1984 [30]. It began with his doctoral student’s studies, i.e. Sapolsky’s and Woessner’s discovery of articular cartilage neutral metalloproteinases (59).
Cartilage, Synovial Proteases Collaborations, 1981-2000
The initial protease studies on autopsied patellas in 1974 (59) moved to studies of human articular cartilage from surgery that used a modified method to register collagenase, developed by Johanne Martel and Jean Pierre Pelletier who took their basic research arthritis training with Drs. Howell’s proetoglycan group and Dr. Woessner’s protease group. A 1981 paper on collagenolytic activity showed most collagenolysis at the center of osteoarthritic lesions, less in the rim, and still less in remote sites (92), an important follow-up on Erlich’s original discovery of collagenase in osteoarthritic cartilage. A 1983 paper on collagenase and collagenolytic activity recorded high activity in and around the edge of osteoarthritic erosions, less activity back from the edge, and little or no activity in distant locations (99). A study in completely controlled conditions in the Pond-Nuki dog model of osteoarthritis clearly showed the progression of lesions (100).
When the Pelletier collagenase assay was adapted to proteoglycanase assay, there was a 3-10 fold elevation of active proteoglycanase activity and, as with collagenase, highest enzyme levels were in the center of osteoarthritic erosions (107). Data strongly supported the role of neutral metalloproteoglycan-degrading enzyme in the destruction of rheumatoid human arthritis cartilage (110), and, as with the collagenase study (100), studies on collagenase in the closely controlled dog model were similar to human findings but were more sharply distinguished (111).
Almost two decades of steadily improved assay methods developed by Woessner, Dean, et al permitted evidence for metalloproteinase and metalloproteinase inhibitor imbalance in human osteoarthritic cartilage to be elucidated. In mild to moderate osteoarthritic lesions, best studied in medial plateau samples and rigorously handled to prevent artifacts, levels of tissue inhibitor of metalloproteinase (TIMP) increased 50% versus controls, whereas acid and neutral protease activity increased 150%, with best results with regard to localization and Mankin score from the acid metalloproteinase (133). The collaborative papers with the Pelletiers and Woessner, as well as those under Dr. Woessner’s aegis which followed, indicated that the metalloproteinases, and particularly stromelysin and TIMP, have involvement in osteoarthritis degradation in humans, dogs, and rabbit models of osteoarthritis. Dr. Herman Cheung was invited by Dr. Howell to take over all laboratory directing functions in 1997 - all work on BCP and MMP-13 below.
The collaborative cartilage and synovial protease studies begun in 1980 continued into the current millennium. Another study in Pond-Nuki animals demonstrated that a peptide, shown by Clemmons to inhibit action of the canine insulin-growth factor (IGF) binding protein 5, inhibits IGF binding protein proteolysis in articular cartilage and joint fluid and is thereby associated with improved osteoarthritis (177). Basic calcium phosphate (BCP) crystals were shown to up-regulate metalloproteinases, but down-regulate TIMP in human fibroblasts. Since phosphocitrate specifically blocks this differential reaction, key pathways in the action of BCP may be their action in the pathogenesis of osteoarthritis (176). Similarly, since human matrix metalloproteinase 13 (hMMP-13) is down-regulated by wild type p53, the gene expression of hMMP-13 could be dysregulated during the disease progression of arthritis and other diseases associated with p53 inactivation (172).
Proteases in Cell Culture Collaborations, 1977-1986
A 1977 review of the status of protease, during a period of intense efforts to separate and identify the individual enzymes that contribute to neutral pH degradation of identifying substrates, also described directions for future research (76). In a series of papers with Malemud, Moskowitz, Sapolsky, Howell and others which followed, metal-dependent proteases were elaborated almost completely into the media in chondrocyte culture, suggesting their degradative role in extracellular cartilage matrix at a time when many involved scientists were skeptical that extracellular neutral metalloproteases would become important to human physiology or disease [See paper of Struglics et al. and editorial by Sandy in ref 28].
Metal-dependent neutral proteoglycanase activity from the cytoplasm of lapine articular chondrocytes, partially inhibited by orthophenanthroline and EDTA (ethylenediaminetetraacetic acid), were synthesized and secreted into the culture medium where they degraded proteoglycans (85). A latent collagenase that degrades human cartilage type II collagen was found to be secreted from secondary spinner and monolayer cultures of lapine articular chondrocytes. A latent form was secreted since activity was heightened by preincubation with either trypsin or APMA (88). Inhibitory activity on the proteoglycanase included data on the pH optimum of the proteoglycanase and of the heat labile inhibitor (89).
An important part of the early work on the detection of heat labile inhibitors from cultured articular chondrocytes, that contributed to the discovery of TIMP, was also led by leaders in the field from Case Western Reserve University and involved labs in Rhode Island and Miami (93,94). A 35,000 mw inhibitor from bound metalloprotease was released and found to inhibit uterine collagenase and gelatinase, but not clostridial collagenase (98). A factor derived from activated rabbit macrophases was found to cause ascorbic acid to stimulate the resorption of canine articular cartilage (112). The collaborative work on proteases in cell culture and cartilage matrix degradation was published in 1986 (119).
Pan-Protease Inhibitorial Agents in Treatment of Osteoarthritis Animal Models, 1986-1994
Attempts at conservation of cartilage in animal models of osteoarthritis by Howell, Altman and Dean included some failures, but were largely successful.
That glycosaminoglycan polysulfate (GAGPS) retarded development of osteoarthritic type erosions, both prophylactically and therapeutically, and favorably influenced the histological severity of lesions was demonstrated in the Moskowitz lapine model, which included a partial medial meniscectomy (116). Parenteral GAGPS was shown to reduce to almost normal levels the elevated neutral metalloproteinase activity in positive controls (118). In the canine model, GAGPS reduced saline-induced swelling to almost normal levels and significantly reduced collagenase activity (122).
GAGPS administered prophylactically for four weeks reduced all morphological parameters of an osteoarthritis response in a canine model, but neutral proteases changes were not significant (128). GAGPS administered therapeutically for 12 weeks significantly reduced the high levels of proteoglycanase and restored significant swelling properties to normal in a canine model (131).
Powerful, positive chondroprotective responses of glycosaminoglycan-peptide association complex (Rumalon) in the Moskowitz lapine model included up-regulation of TIMP, synthesis of collagen, and suppression of elevated proteoglycanase (137). A further study also showed chondroprotection by Rumalon in the lapine model (145).
The biochemical and histological protection against cartilage breakdown by prophylactic administration of tiaprofenic acid was demonstrated in the Pond-Nuki canine model (141). Therapeutic treatment of osteoarthritis in the canine model with tiaprofenic acid and indomethacin for 12 weeks resulted in the retention of fast sedimenting aggregates of proteoglycan at about normal control levels by arresting their breakdown and loss (144).
The most complete suppression of neutral metalloproteoglycanase and elevation of TIMP obtained were achieved by insulin-growth factor-1 (IGF-1) and sodium pentosan polysulfate combination therapy (150, 153) in a canine model. In a review of experimental approaches to chondroprotection, combination therapy with a protease inhibitor and an appropriate growth factor seemed the most promising (151). However, an inventory of uncertainties mounted as the complexity of degradation was unraveled (155) in 1999 with the discovery of aggrecanases.
Clinical research collaborators - The well known gout researcher and editor of JAMA, John Talbott, who moved to Key Biscayne in 1968, joined and stayed with Dr. Howell's unit for his remaining 22 years. Partly at his own expense he started and edited a new journal Seminars in Arthritis and Rheumatism which is still highly rated in its 4th decade of publication. Roy Altman, Professor of Medicine who recently relocated to UCLA, was effective in his academic outreach and invaluable to me for his administrative services, production of animal models and his related research. Harvey Brown's leadership, clinical teaching and patient care were kept in high esteem. Norman Gottlieb was gifted at bedside teaching and known for his studies on gold pharmacology and help on the journal Seminars. Elaine Tozman and Duane Schultz, a research immunologist who joined her to teach the sophomore Mechanism of Disease Course, were and still are stars. Carlos Lozoda has added strength from his interest and NYU training in inflammatory arthritides and fine tuning patient management. These colleagues raised clinical management and teaching standards.
Basic research collaborators - In the early 1960s optimism for much expanding research funding from NIH was in the air as some experienced scientists in the early waves of Cuban refugees reached Miami. From the faculty at U. Villanova and U. Havana came an academic leader in physical biochemistry, Julio Pita, who helped build a program around miniaturized analytical chemical methods. The new methods would be applied to study fluid phases of mineralizing bone or cartilage. I am grateful to him for becoming the leading basic scientist for our laboratory and source of dedicated, skillful biochemical technicians from Havana. Pita’s primary assistant was Francisco Muller,a physicist and professional musician who would spellbind visiting professors with his pianoplaying. A colleague, Fred Woessner and his research team in the Biochemistry Department, settled their research efforts on connective tissue proteases. The outstanding developments made by Woessner on analyzing types of proteases, especially matrix metalloproteases, and Pita's advances in analytical chemistry relevant to cartilage proteoglycans and electrolytes enabled a program of diverse osteoarthritis oriented research, with a competitive edge, to grow over the next three decades. Herman Cheung, after my retirement in 1995, some time soon thereafter took over effectively running the laboratory program.
Extramural collaborators -A steady stream of visiting lecturers in our fields and that of Dr. Woessner were organized by myself and Altman and paid from our mutual differing funds, the most important of which was the annual Kroc Foundation lectureship in later years. By 1995, 90 papers resulted from our collaborations at 21 different centers. The longest and most productive of papers were on proteases in cartilage with Fred Woessner and associates in the Biochemistry Department at home, proteases in cultured cartilage cells with Charles Malamud and associates at Case Western Reserve, biomechanical studies of human knee cartilage with Van Mow and associates at Columbia University and later Setton and Guilak at Duke, and biochemistry of growth plate cartilage with Barbara Boyan, Zvi Schwartz and their team at University of Texas, San Antonio, as well as Klaus Kuettner and Eugene Thonar at the Biochemistry Department of Rush Medical Center. I am indebted to these and many other collaborators for their high quality contributions.
Students - Since 1970, I kept space in the lab for qualified students to produce work counting toward predoctoral, doctoral, and post doctoral training in cartilage pathophysiology. Those at an appropriate stage became senior authors on important papers, and some progressed to become important leaders in cartilage and osteoarthritis research. I was a mentor to these bright individuals and helped them make decisions on where to go next in the research market place. Dr. Woessner developed a well known and highly sought predoctoral program leading to a PhD and a career in molecular biochemistry of proteases, as well as post doctoral training in this research. The Howell-Pita program focused on closely guided technicians and always two to three postdoctoral students and an experienced researcher on sabbatical. See my bibliography for papers involved. Dan Dantini spent two years in the electrolyte area, and went on to do research at the University of Pittsburgh’s ENT Dept. Leon A. Cuervo spent five years with the lab. Already experienced in pharmacology, he went on to become chairman of the Biology Department at Florida International University. David R. Simon, trained by Irwin Berman, went on to the Electron Microscopy Unit at the National Naval Medical Center, Bethesda. He did his main work on healing rickets and matrix vesicle mineralization. Asher I. Sapolsky received his PhD and post doc training from Woessner. He went on to the Miriam Hospital Laboratory in Providence, RI and developed collaborations with leading osteoarthritis researchers at Case Western Reserve, Charles Malamud: Roland Moskowitz and Victor Goldberg. He engendered a prominent scientific career after age 55 based on many well-founded chondrocytic cell culture protease and protease inhibitor studies. Teresa Morales received her PhD from Woessner and her early post doc training with him as well as later, Vincent Hascall and Klaus Kuettner. She was taken into the Massachussets General Hospital Orthopedic Department where she has a thriving lab and is teaching Harvard medical students. She is senior author of classic studies on proteoglycans and cartilage physiology. Jean Pierre Pelletier and Joan Martel-Pelletier, a husband and wife team, came to work in Howell's and Woessner's laboratories on a two year fellowship. I guided a study on chondrocalcinosis with them. The Pelletiers returned to the University of Montreal and I believe created one of the few most advanced osteoarthritis biochemistry groups world wide, with phenomenal productivity in diverse, well conducted basic studies important to osteoarthritis pathogenesis. Daniel H. Manicourt, MD, an experienced researcher, returned home to an advance in his position at Louvaine University Medical Center’s Immunopathology Research Institute and with an award of a PhD. He spent five years in my laboratory where he was a hard benchworker who applied Pita's associative extraction method for removing proteoglycans and studying them for aggrecan aggregate size distribution as affected by hyaluronan and link protein. Productive collaborations were set up with Eugene Thonar at Rush’s Biochemistry Department in the mid 1980s which continue to the present. David D. Dean obtained his PhD at the University of North Carolina at Chapel Hill and his post doctoral fellowship with Woessner in the early 1980s. After my phosphonate rickets paper in 1980, he shifted away from micropuncture work, except in regard to an unpublished preliminary study on the fate of injected microspheres at the tips of growth plate cell columns and their rapid appearance five cells caudad. The fast rate of my mineralizing septae in his rat model seemed to require a flow of mineral ions to speed up production by one or more mineralizing mechanisms. Thus I developed my own theory on fast mammalian growth plate mineralization. With the loss of Gregg, the VA electron microscopist, this project terminated. By 1984, I was able to fund Dean on his phospholipids in mineralization via an NIH grant and allow for the early analysis of enormous upregulation of collagenase followed by organ and cell cultures, all showing insufficient tissue inhibitor of metalloproteinases in studies with Woessner. Dean in the early 1990s moved to San Antonio where he worked with Barbara Boyan on many projects in one of the few most active mineralization laboratories. Dean became director of the Orthopedic Research Laboratory at the University of Texas, San Antonio after Boyan moved to Georgia Tech. Denouement I am grateful to a considerable list of research donors, mature scientists, as well as clinical and basic research trainees, many of whom are named in the bibliography, for important and differing types of contribution to the Department of Medicine, Arthritis Division, at the University of Miami Miller School of Medicine. The Calder Medical Library staff has been of enormous help in creating this report, especially Suzetta Burrows and Jenny Garcia-Barcena.
Editorial Responsibilities: Co-editor, Seminars in Arthritis &Rheumatism, 1984 – 2000 Editorial Board, Arthritis & Rheumatism, 1961-84 Editorial Board, Calcified Tissue International, 1978-84 Editorial Board, Journal of Orthopaedic Research, 1984-90 Editorial Board, European Journal of Rheumatology and Inflammation: OA Series – 1991 Editorial Board, Osteoarthritis and Cartilage Journal, 1993 - 2000
Professional and Honorary Organizations: American Rheumatism Association (now American College of Rheumatology): Member, 1955- Diagnostic Criteria Committee, 1958-60 Membership Committee, 1963-66 (Chairman-final year) Education Program Committee, 1962-68 (Chairman-last two years) Therapeutic Trials Committee, 1963-65 Program Committee, 1970-75, 1978-81, 1985-88 (Second Vice President, 1970) Honorary Membership Committee, 1979-81 (Chairman-final year) Future Sites Committee, 1983-85 (Chairman, 1985)
National Arthritis Foundation: Member, 1955- Fellowship Committee, 1978-1981 (Chairman-final year) Fellowship and Investigator Committee, Member, 1986-89 Florida Arthritis Foundation District Member, 1957- Board, 1975-79 South Florida Division Founding Member, 1957-59 Fund raising campaign, annually through 1975 Numerous lectures and other activities for lay people, 1978-81 Dade County Chapter Founding Member, 1957-59 Fund raising campaign, annually through 1975 President, 1966-68 Program Chairman, 1969-72
Orthopedic Research Society Member, 1970- Program Committee, 1975-79 (Chairman-final year)
Gordon Conference on Bones & Teeth Annual Participant, 1958-1990 Vice Chairman and Chairman, 1972-73
Honors and Awards:
Phi Beta Kappa Bowdoin College, 1944. Markle Foundation Scholarship, 1958-62. Biochemistry and Cell Research Institutes – Nobel Institute, 1960-62 Fellowship. Philip Hench Award for Arthritis Research, 1970, National Society of Military Surgeons. Honorary Member, Hospital for Special Surgery (Cornell), Distinguished Lecturer, 1971. AF National Distinguished Service Award, 1972-73, 78-79. Guest Investigator to French National Institute of Health (INSERM), 1972. Guest Lecturer, Cochin Medical School, Paris, and Lyon and Montpelier Medical Schools. Pemberton Lectureship Award, November, 1973, Philadelphia College of Physicians. Kappa Delta Award, March, 1975, National Orthopaedic Research Award. David S. Howell Study room, Louis Calder Memorial Library, University of Miami, Dedicated January, 1976. AMA Physician’s Recognition Award, 1976. Honorary Doctorate in Medicine, Faculty of Medicine, University of Umea, Sweden, October, 1977. Biological Mineralization Scientific Award, International Dental Research Society, 1978. Richard H. Freyberg Lecturer, Hospital for Special Surgery, 1980. Geigy International Research Prize in Rheumatology, Paris, 1981. (co-winner) Only V.A. Investigator to ever receive four successive 5-year competitive investigator Awards, 1969-93. (and 4 added years). 1993 Central Office VA award. Allied Signal Award, Osteoarthritis award, 1987-90. NIH MERIT Award, 1990-2000 (by Study Section). Designation of Master, in American College of Rheumatology, July 1992. Nomination for the Lee Howley Arthritis Research Prize, June 1992. Nomination for the Arthritis Foundation Help & Hope Award for Excellence in Rheumatology. UM Distinguished Faculty Scholar Award, April 6, 1994. Roussel-OARS International Prize for Osteoarthritis Research, Paris, 1994. Honorary President, 15th European Osteoarthrology Symposium, Ghent, Belgium, 1996. Honored along with peers Fujio Suzuki, Department of Biochemistry, Osaka Dental School and Yousef Ali, National Orthopedic Hospital, University of London, Pathology Department, for our life work on the growth plates, 1st International Symposium on Mineralization of Growth Plate Biology, 2002. Osteoarthritis Research Society International Lifetime Achievement Award, 2005. Other Honors:
Argentinian Rheumatology Society, Honorary Lecturer, 1977. Philippine Rheumatology, Group Solicitor, 1975-76 (along with John Talbott). British Bone and Tooth Society, Honorary Lecturer, Oxford, 1970s.
Community Activities: United Health Foundation, Miami-Dade County Board of Directors, 1966-80 Chairman, Scientific Advisory Committee, 1967-68 Easter Seal Society, Medical Advisory Committee, 1974-95 Planned Parenthood Society of Dade County Chapter, 1987- American Art League, Dade County Chapter, International Society of Marine Painters, 1986 – present Arthritis Foundation, State Executive Committee (Florida Chapter), 1981-91 AF Biomedical Research Center Grant, 1987-90
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