Fred Harwood

University of California, San Diego, San Diego, California, United States

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Publications (16)29.46 Total impact

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    ABSTRACT: Biglycan, a small leucine-rich proteoglycan, has been shown to interact with extracellular matrix (ECM) collagen and may influence fibrillogenesis. We hypothesized that biglycan contributes to post-myocardial infarction (MI) scar development and that the absence of biglycan would result in altered scar structure and mechanics. Anterior MI was induced in biglycan hemizygous null and wild-type mice by permanent ligation of the left coronary artery. The initial extent of ischemic injury was similar in the two groups, as was the infarct size after 30 days, although there was some tendency toward reduced expansion in the biglycan-null. Electron microscopy revealed that collagen fibrils had a smaller average diameter and a narrower range in the biglycan-null scar, as well as appearing more densely packed. In vivo strain analysis showed that biglycan-null scars were stiffer than the wild-type. Remote LV collagen concentration tended to be reduced in biglycan-null hearts, but the difference was not statistically significant. Null-expression of biglycan may alter collagen fibril ultrastructure, and thereby influence scar mechanics and remodeling.
    Molecular & cellular biomechanics: MCB 03/2008; 5(1):27-35.
  • Arthroscopy The Journal of Arthroscopic and Related Surgery 06/2007; 23(6):e36-e37. · 3.19 Impact Factor
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    ABSTRACT: Growth factor delivery may be useful to accelerate the rate of tendon healing. Before in vivo use, however, the effects of growth factors on tendon cells need to be well characterized. The purpose of this study was to evaluate the effects of 4 growth factors on intrasynovial tendon fibroblast proliferation and collagen production in vitro. Our first hypothesis was that platelet-derived growth factor BB (PDGF-BB) and basic fibroblast growth factor (bFGF) would promote cell proliferation and collagen production. Our second hypothesis was that there would be a positive effect from the combination of PDGF-BB and bFGF. The growth factors PDGF-BB, bFGF, vascular endothelial growth factor (VEGF), and bone morphogenetic protein 2 (BMP-2) were evaluated in vitro with canine flexor tendon fibroblasts. The effects of single factors (PDGF-BB, bFGF, VEGF, or BMP-2) or a combination of factors (PDGF-BB and bFGF) on cell proliferation (ie, thymidine incorporation) and collagen production (ie, proline incorporation) were evaluated. The results supported our hypotheses. Cell proliferation increased significantly with PDGF-BB and bFGF. Collagen production also increased significantly with PDGF-BB and bFGF. Cell proliferation and collagen production were unchanged with VEGF and BMP-2. A dose-response effect was seen for PDGF-BB combined with bFGF. The combination of PDGF-BB and bFGF led to an increase in cell proliferation but no change in collagen production compared with each factor alone. The growth factors PDGF-BB and bFGF significantly increased flexor tendon fibroblast proliferation and matrix synthesis when applied singly. Administration of PDGF-BB and bFGF combined led to increased proliferation to single factors.
    The Journal Of Hand Surgery 06/2005; 30(3):441-7. · 1.66 Impact Factor
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    ABSTRACT: To investigate rates of cellular proliferation and matrix turnover in autogenous flexor tendon grafts, hindlimb intrasynovial (flexor digitorum profundus) and extrasynovial (peroneus longus) tendons were placed within the synovial sheaths of the medial and lateral forepaw digits of 18 dogs and treated with controlled early passive motion. After the dogs had been killed, short-term culture and labeling in vitro were utilized to determine rates of DNA, proteoglycan, collagen, and noncollagen protein synthesis. Schiff base covalent collagen crosslink concentrations and total collagen and protein content also were evaluated at intervals through 6 weeks. Tendon grafts of extrasynovial origin showed greater rates of DNA synthesis and significantly elevated levels of proteoglycan, collagen, and noncollagen protein synthesis and Schiff base covalent collagen crosslink concentrations (dihydroxylysinonorleucine) compared with intrasynovial tendon grafts. It was not clear to what extent the increased activity in the extrasynovial graft was due to actual differences between the intrasynovial and extrasynovial tendons or to the responses of the connective tissue surrounding the extrasynovial tendon graft. Since both types of grafts demonstrated similar unaltered levels of collagen and protein content over time, these data suggest greater rates of matrix turnover in tendon grafts of extrasynovial origin than in those of intrasynovial origin. Coupled with previous findings showing increased cellular proliferation in extrasynovial tendon grafts, these data indicate that the process of translation to an intrasynovial environment necessitates a more active process of soft-tissue repair and remodeling when extrasynovial donor tendons are used.
    Journal of Orthopaedic Research 02/2005; 13(1):58 - 66. · 2.88 Impact Factor
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    ABSTRACT: In vivo animal studies have indicated that the complex structure of the tendon-bone interface may not be restored after repair even under optimal conditions. Controversy exists about the histologic findings in the early postoperative period after tendon reattachment to bone; this may have impact on biomechanical properties. The objective was to study the histologic structure and immunohistochemical staining of the tendon-bone interface in a large model of digital flexor tendon-bone repair. The hypothesis was that the tendon-bone interface matures and assumes a progressively more anatomic histologic and immunohistochemical appearance during the first 6 weeks after repair. Twenty-four canine flexor digitorum profundus tendons were released from their insertion by sharp dissection and repaired to bone. The forelimb was immobilized after surgery and 10 minutes of daily passive motion rehabilitation was performed. Dogs were killed at 10, 21, and 42 days after surgery. Hematoxylin-eosin and immunohistochemical staining for types I, II, and II collagen were performed. Although at both 10 and 21 days after surgery substantial inflammation was seen at the tendon-bone repair site, this had decreased markedly by 42 days. Although direct apposition of tendon to bone was seen at 42 days, the mature tendon-bone insertion site was not recreated by this time. Staining for types I and III collagen was diffuse throughout the tendon-bone insertion throughout the interval examined. These findings suggest that at 6 weeks after surgery the intact tendon-bone repair site shows minimal histologic and molecular similarity when compared with unoperated specimens.
    The Journal Of Hand Surgery 06/2003; 28(3):469-74. · 1.66 Impact Factor
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    ABSTRACT: The biochemical means by which accelerated rehabilitation alters intrasynovial flexor tendon repair site collagen synthesis and extracellular matrix maturation are not fully understood. We hypothesized that an increased level of applied rehabilitative force in a clinically relevant animal model would hasten the maturation of the repair site extracellular matrix as demonstrated by total collagen and collagen cross-link assessment. Twenty-eight flexor digitorum profundus tendons from 14 adult dogs were transected and repaired. The animals received either low- or high-force rehabilitation and were killed 10, 21, and 42 days after surgery. A 10-mm segment of tendon surrounding the repair site was obtained. Biochemical analysis showed that total collagen concentration was significantly reduced at each time point, that the reducible cross-link ratio of dihydroxylysinonorleucine to hydroxylysinonorleucine was significantly increased at each time point, and that the nonreducible pyridinoline cross-link content was significantly decreased at 10 days in both rehabilitative groups. Total collagen content did not vary to a statistically significant degree with either time or as a function of rehabilitation type. Based on these findings several clinically relevant observations can be made. Increasing collagen concentration and repair site maturation do not explain the previously demonstrated increased tensile properties of tendon that occur between 3 and 6 weeks after repair. Higher force rehabilitation does not alter the biochemical composition of the healing tendon through 6 weeks. Coupled with other recent data these findings suggest that high-force rehabilitation does not stimulate accelerated healing after intrasynovial flexor tendon repair.
    The Journal Of Hand Surgery 10/2001; 26(5):841-6. · 1.66 Impact Factor
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    ABSTRACT: The effects of beta-aminopropionitrile, a known inhibitor of lysyl oxidase, on the extractability of newly synthesized collagen and integrative cartilage repair were determined in explant cultures of adult bovine articular cartilage. Dose-escalation studies indicated that treatment of cartilage explants for 6 days with beta-aminopropionitrile caused a dose-dependent inhibition of proteoglycan synthesis ([35S]sulfate incorporation) with a 50% inhibition at 2.2 mM. However, 0.25 mM beta-aminopropionitrile had no detectable effect on proteoglycan synthesis and was thus used for subsequent experiments. Treatment of cartilage with beta-aminopropionitrile for 14 days increased the extractability of newly synthesized collagen with 4 M guanidine-HCl while having little effect on proteoglycan synthesis, proteoglycan deposition, collagen synthesis (formation of [3H]hydroxyproline after labeling with [3H]proline), collagen deposition, or cartilage cellularity (DNA content). In untreated cultures, the percentage of radiolabeled collagen ([3H]hydroxyproline) that was extractable after 1 day of radiolabeling, 6 days of radiolabeling, or 6 days of label and 6 days of chase decreased from 81 to 25 and 9%, respectively. In beta-aminopropionitrile-treated cultures, the extractability was relatively higher (96, 62, and 47%, respectively). Treatment with beta-aminopropionitrile after radiolabeling with [14C]lysine also significantly inhibited the formation of the reducible crosslink [14C]dihydroxylysinonorleucine without affecting the overall deposition in cartilage of [14C]lysine and [14C]hydroxylysine. In functional repair studies, treatment with beta-aminopropionitrile caused an almost complete inhibition of integration between pairs of cartilage explants maintained in apposition for 2 weeks. These results indicate that beta-aminopropionitrile blocks the formation of collagen crosslinks in cartilage explants and suggest that such crosslinks are critical to integrative cartilage repair.
    Journal of Orthopaedic Research 12/1999; 17(6):850-7. · 2.97 Impact Factor
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    ABSTRACT: This study assessed a fresh medial meniscal allograft transplantation model in the rabbit knee. Biological characterization included assessment of collagen remodeling of the allograft and the potential protection against cartilage degeneration. Allograft transplantation was performed on the left knee, and total meniscectomy on the right knee. Forty-seven rabbits were operated on and assessed at 9, 12, and 26 weeks. Fresh medial meniscal allografts showed collagen remodeling that paralleled the revascularization and cellular proliferation of the allografts. Revascularization was shown as early as 9 weeks from the periphery, extending to the inner one-third of the allograft by 26 weeks. Viability assessment of the meniscal allograft cells showed live cells at the periphery of the allograft at 9 and 12 weeks. At time 0, i.e., the time of the transplant, few viable cells were observed within the donor tissues. Biochemically, collagen remodeling, in terms of increased reducible collagen crosslinks, i.e., dihydroxylysinonorleucine, and the percentage of collagen present, was seen throughout the 26-week observation period. At 26 weeks, the meniscal allografts inhibited degenerative changes of the femoral and tibial cartialage compared to results with total meniscectomy.
    Journal of Orthopaedic Science 05/1997; 2(3):171-179. · 1.01 Impact Factor
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    ABSTRACT: To determine the precise mechanism by which contact tendon healing occurs at the cellular level, the production of pro alpha (I) collagen messenger RNA (mRNA) produced by fibroblasts of healing intrasynovial flexor tendons was determined by an in situ hybridization technique. The repair site and the proximal and distal tendon stumps of repaired tendons treated with early controlled passive mobilization were fixed and buffered in formalin, 3, 7, 10, and 17 days after repair. A complimentary DNA (cDNA) probe corresponding to alpha (I) procollagen mRNA was labeled with [32P]d-CTP. After hybridization, autoradiography, and staining of the sections, the level of procollagen mRNA was assessed by microscopic examination. Rising levels of procollagen mRNA, indicating progressively increasing levels of synthetic collagen activity, were detected in the healing tendons through 10 days. A moderate decrease in procollagen mRNA was seen at 17 days. Genetic expression for procollagen mRNA was localized specifically to the epitenon cells on the tendon surface overlying the repair site and to cells in the gap between the tendon stumps. No detectable expression was noted in endotenon fibroblasts. The finding of high levels of expression for procollagen type I mRNA in the surface layer of healing tendons demonstrates that cells intrinsic to tendon epitenon contribute the greatest quantity of native tendon collagen to the repair site during these important early intervals after tendon suture.
    The Journal Of Hand Surgery 06/1992; 17(3):551-8. · 1.66 Impact Factor
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    ABSTRACT: The purpose of this study was to assess, morphologically and biochemically, the effect of hyaluronan (HA) on the early repair process of the anterior cruciate ligament (ACL). Following partial bilateral laceration in the midsubstance of the cruciate ligament, a single dose of HA (MW of 3.6 x 10(6] was injected in one knee and saline in the contralateral knee. Postsurgery, the rabbits were allowed normal (nonimmobilized) cage activity, and were killed after 4 (n = 11) and 12 (n = 10) weeks. The ligaments were evaluated by gross morphology and graded according to the degree of repair. We used grades 1,2, and 3 for uncovered, partially covered, and totally covered lacerations, respectively. Five of the HA-treated ligaments at each time studied were completely covered, compared to 0 at 4 weeks, and 1 at 12 weeks in the saline group. Paired evaluations of the lacerated ACLs showed that the HA-treated ligaments received a healing grade higher than the ligaments exposed to saline in 14 of the 21 animals. In the remaining animals, there was no difference between the sides. The repaired tissue of the ACLs was also examined by light and electron microscopy. When compared qualitatively with saline controls, HA-treated ligaments exhibited a more pronounced repair, with an increased angiogenesis and less inflammatory response. Biochemical analysis demonstrated a mean higher value of type III collagen in the HA-treated injured ACL than in saline-treated injured ACL (13.4 +/- 1.1% and 11.0 +/- 0.8%, respectively). This increased synthesis of type III collagen in the HA-treated injured ACL was statistically higher (p less than 0.05) when compared to the saline-treated injured ACL.
    Journal of Orthopaedic Research 06/1990; 8(3):425-34. · 2.97 Impact Factor
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    ABSTRACT: A rabbit model for anterior cruciate ligament (ACL) reconstruction using autogenous patellar tendon was utilized to study the early events of autograft cellular dynamics. Biochemical, autoradiographic, histological, and vascular injection techniques demonstrated that the native autograft cell population rapidly necroses. This repopulation occurs without a vascular contribution; cells entering the autograft are reliant upon synovial fluid nutrition.
    Journal of Orthopaedic Research 02/1989; 7(2):235-42. · 2.97 Impact Factor
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    ABSTRACT: Fresh osteochondral allografts were stored at 4 degrees C in tissue culture media at variable time periods (3, 7, 14 and 28 days). Sterilely dissected tibial plateaus with a standardized 1/2 cm subchondral bone "shell" were obtained from canines 1-3 hrs post mortem. X-rays were taken to determine maturity of the animals. Only mature animals (closed epiphyses) were considered for the study. Histologically, safranin 0 (metachromatic stain for glycosaminoglycans) was observed in all experimental specimens. H&E stained sections showed at all time periods of 3, 7, 14 and 28 days that the cell morphology and arrangements were similar in the superficial and deep areas of the cartilage obtained from the stored osteochondral allograft when compared to the control articular cartilage. The cells were in lacunae and arranged in clusters. Biochemically, glycosaminoglycans and collagen content showed no difference at the 95% level of confidence during the duration of the study (28 days) when compared to the 0 day control cartilage. Collagen typing, based on the assessment by HPLC of the CNBr peptides showed the major presence of type II collagen (no evidence of dedifferentiation was observed). No type I was found to be present. Some apparent variations in the proportions of minor collagen components were noted--e.g. at 14 days the cartilage appeared to contain increased amounts of type XI but little or no type IX collagen (HMW, LMW) when compared to the day 0 control. At 28 days a shift to a larger amount of type IX collagen occurs, especially in the LMW component, with a small amount of type XI collagen when compared to normal day 0 articular cartilage. Cell viability, i.e., the ability of the allograft tissue to incorporate 35SO4 in the synthesis of glycosaminoglycans, was intact up to 28 days of storage.
    Connective Tissue Research 01/1989; 23(1):89-99. · 1.98 Impact Factor
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    ABSTRACT: The reconstructed anterior cruciate ligament was studied in the rabbit using the medial third of the patellar tendon. Tritiated proline, 100 microCi/kg body weight, was injected intra-articularly to insure detection of the metabolic conversion product 3H-hydroxyproline in the avascular graft. During the immediate postoperative period, nutrients were found to derive from the synovial fluid through a process of diffusion, demonstrating that synovial nutrition occurs prior to revascularization of the graft.
    Acta Orthopaedica Scandinavica 07/1986; 57(3):201-3.
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    ABSTRACT: A method for estimating type II to type I collagen ratios in small tissue samples has been developed. The cyanogen bromide peptides of the tissue collagens were analyzed by SDS-gel electrophoresis. Marker peptides representative of each collagen type were established and their relative amounts determined by integration of the stained peptide bands following gel scans. Marker peptide ratios were then computed for each of several standard type II/type I mixtures and these peptide ratios were mathematically correlated with the corresponding type II/type I collagen ratios. A linear relationship between marker peptide ratio and collagen type ratio was established. This relationship was applied to the analyses of type II/type I ratios in samples of rib perichondrium and neocartilage derived from perichondrial graft repairs of full thickness femoral condyle defects. The results indicated that perichondrial grafts synthesize both types II and I collagens and that the proportion of type II increases with increasing post-transplant time.
    Collagen and related research 10/1985; 5(4):337-47.
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    ABSTRACT: Joint stiffness secondary to immobilization was inhibited by intra-articular hyaluronic acid injection in an experimental joint contracture in rabbits. Biochemical and biomechanical parameters were used to evaluate the joint stiffness after nine weeks of immobilization. In all treatments, hyaluronic acid reduced the measured stiffness in the contracture by approximately 50% as compared to the contractures of the untreated rabbits. In addition, hyaluronic acid prevented the loss of glycosaminoglycans (GAGs) (as measured by hexosamine), which normally occurs in untreated contractures. The results are related to a working hypothesis that intra-articular injections of drugs such as hyaluronic acid (Healon-R) will stimulate hyaluronic acid synthesis within the matrices of periarticular connective tissue (PCT). If the spacing and lubricating properties of the glycosaminoglycans could be maintained in the stress-deprived state, the "centripetal collapse" of the fibrillar matrix could be avoided, anomalous cross-links could be minimized, and more normal joint mechanics could be retained.
    Clinical Orthopaedics and Related Research 07/1985; · 2.88 Impact Factor
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    ABSTRACT: Rabbits immobilized with one leg pinned in a fully flexed position, were treated with 60 mg of D-penicillamine/kg three times a week for 9 weeks. After this period of immobilization the animals were sacrificed and the periarticular connective tissue collagen reduced with [3H] NaBH4 for determination of collagen cross-links. There was a significant decrease in the formation of cross-links in the control and immobilized knee in the treated animals as compared to the untreated animals. Penicillamine also blocked the increase in DHLNL, HLNL and HHMD previously demonstrated in immobilized rabbit knees. This is the first demonstration that in vivo administration of penicillamine directly inhibits cross-linking reactions in collagen.
    Connective Tissue Research 02/1977; 5(3):179-83. · 1.98 Impact Factor

Publication Stats

430 Citations
29.46 Total Impact Points

Institutions

  • 1985–2005
    • University of California, San Diego
      • • Department of Bioengineering
      • • Department of Medicine
      • • Department of Orthopaedic Surgery
      San Diego, California, United States
  • 1992
    • Massachusetts General Hospital
      • Department of Orthopaedic Surgery
      Boston, Massachusetts, United States
  • 1986
    • National University (California)
      San Diego, California, United States