Hung Pham - Academia.edu (original) (raw)
Papers by Hung Pham
Journal of Hand Surgery-american Volume, 2004
Purpose: Postoperative adhesions frequently compromise the success of flexor tendon repair. Manip... more Purpose: Postoperative adhesions frequently compromise the success of flexor tendon repair. Manipulation of growth factors responsible for scar formation may be a method of decreasing adhesion formation. Transforming growth factor beta (TGF-) is a key cytokine in the pathogenesis of tissue fibrosis. The purpose of this study was to examine the effectiveness of TGF- neutralizing antibody in blocking TGF--induced collagen I production in rabbit flexor tendons in vitro. Methods: Sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were obtained from rabbit flexor tendons. Each cell culture was supplemented with 1 ng/mL of TGF- along with increasing doses of TGF- neutralizing antibody (0.1-2.0 g/mL). Collagen I production was measured by enzyme-linked immunoabsorbent assay and TGF- bioactivity was measured by the luciferase assay. Results were compared with TGF- alone and unsupplemented controls. Results: The addition of neutralizing antibody significantly reduced TGF--induced collagen I production in a dose-dependent manner in all 3 cell cultures. TGF- bioactivity was also reduced by its neutralizing antibody. Conclusions: This study shows that TGF- inhibition through its neutralizing antibody was effective in cultured flexor tendon cells. The results encourage further experiments that use such agents to modulate flexor tendon wound healing in in vivo models in the hope of eventually blocking the effect of TGF- on flexor tendons clinically. (J Hand Surg 2004;29A:230 -235.
Journal of Hand Surgery-american Volume, 2002
Flexor tendon healing is complicated by adhesions to the surrounding sheath. Transforming growth ... more Flexor tendon healing is complicated by adhesions to the surrounding sheath. Transforming growth factor beta (TGF-beta) is a cytokine with numerous activities related to wound healing. We examined the effects of TGF-beta-1, -2 and -3 on tendon cell proliferation and collagen production. Three separate cell lines--sheath fibroblasts, epitenon and endotenon tenocytes--were isolated from rabbit flexor tendons and cultured separately. Cell culture media was supplemented with 1 or 5 ng/mL of TGF-beta-1, -2, or -3. Cell number and collagen I and III production were measured and compared with unsupplemented control cultures. The addition of TGF-beta to cell culture media resulted in a decrease in cell number in all 3 lines that did not reach statistical significance. There was a significant increase (p <.05) in collagen I and III production with the addition of all 3 TGF-beta isoforms. Modulation of TGF-beta production may provide a mechanism to modulate adhesion formation clinically.
Tissue Engineering, 2006
A significant problem in flexor tendon repair is the lack of suitable graft material for reconstr... more A significant problem in flexor tendon repair is the lack of suitable graft material for reconstruction. The ex vivo production of flexor tendon graft constructs requires the expansion of primary cells. Growth factors, such as platelet-derived growth factor-BB (PDGF-BB), insulin-like growth factor-1 (IGF-1), and basic fibroblast growth factor (bFGF), are known to promote tendon healing and tendon cell proliferation. The purpose of these experiments was to optimize tenocyte proliferation in 3 tendon cell populations using growth factor supplementation. Cells of the synovial sheath, epitenon, and endotenon were isolated from rabbit flexor digitorum profundus tendons and maintained in culture. Cell cultures were supplemented with IGF-1, PDGF-BB, and bFGF alone and in combination. The conditions used for individual growth factor supplementation were IGF-1 (10, 50, and 100 ng/mL), PDGF-BB (1, 10, and 50 ng /mL), and bFGF (0.5, 1, and 5 ng/mL). The conditions used for combinations of growth factors were IGF-1 + PDGF-BB (50 + 10 and 100 + 50 ng /mL, respectively) and IGF-1 + PDGF-BB + bFGF (50 + 10 + 1; 50 + 10 + 5; 100 + 50 + 1; and 100 + 50 + 5 ng/mL, respectively). For all 3 tendon cell populations, proliferation at 72 h was greater in the presence of individual growth factors as compared to controls. With PDGF-BB (50 ng/mL) supplementation, mean absorbance values increased 97% (0.57 to 1.13) in S cells, 37% (0.51 to 0.70) in E cells, and 33% (0.33 to 0.44) in T cells ( p < 0.001). In addition, a synergistic effect was observed. The combination of growth factors resulted in greater proliferation as compared to maximal doses of individual growth factors. In cultures supplemented with IGF-1 (100 ng /mL) + PDGF-BB (50 ng/mL), mean absorbance increased 114% (0.57 to 1.22) in S cells, 63% (0.51 to 0.831) in E cells, and 47% (0.33 to 0.48) in T cells ( p < 0.001). IGF-1 (100 ng /mL) + PDGF-BB (50 ng/mL) + bFGF (5 ng/mL) resulted in the greatest amount of cell proliferation for all 3 tendon cell populations. The mean absorbances increased 251% in S cells, 98% in E cells, and 106% in T cells ( p < 0.001). In summary, IGF-1, PDGF-BB, and bFGF can be used in combination to maximize tenocyte proliferation. Synergism among growth factors may provide a means to facilitate tendon engineering.
Journal of Hand Surgery-american Volume, 2006
Purpose: Dupuytren's disease (DD) is characterized by fibroblastic proliferation of the palmar fa... more Purpose: Dupuytren's disease (DD) is characterized by fibroblastic proliferation of the palmar fascia, often leading to flexion contracture in the hand. Although there is a strong genetic component the genome-wide expression of novel genes is not known. The purpose of this study was to use DNA microarray technology to identify upregulated genes in DD. Methods: Human tissue samples were harvested from 3 patient sources: DD cord tissue (n ϭ 20), normal-appearing adjacent control fascia (n ϭ 15), and palmar fascia from patients having carpal tunnel release (n ϭ 15). DNA microarray analysis was performed on amplified sample RNA. Novel genes were compared with known gene functions. A candidate gene of interest was studied further by using immunohistochemistry on DD tissue samples and controls. Results: Several novel genes not described previously in the study of DD were upregulated significantly, including MafB, collagen type V, ␣-2 (COL5A2), collagen type VIII, ␣-1 (COL8A1), contactin I (CNTN1), and leucine-rich repeat containing 17 (LRRC17). These upregulated genes were compared with their known gene-expression profiles in other tissues and their purported functions. MafB was found to be of particular interest because of its prominent role in tissue development and cellular differentiation. MafB immunohistochemistry showed positive staining in 50% of the DD specimens but complete absence of MafB in all control tissues (adjacent control fascia, carpal tunnel fascia). Co-localization experiments with MafB and ␣-smooth muscle actin showed staining properties in similar regions but these 2 proteins were not confined solely to the same cells. Conclusions: Microarray analysis of DD tissue has identified significant upregulated gene expression of MafB. MafB protein also is found in Dupuytren's cords but not in control fascia. Co-localization data suggest that the association of MafB with DD is not related exclusively to myofibroblast proliferation. Because of its role in fibroblastic transformation in other models MafB and its relationship to the pathogenesis of DD deserves further study. (J Hand Surg 2006;31A: 211-218.
Journal of Hand Surgery-american Volume, 2001
Flexor tendon repair in zone II is complicated by adhesions to the surrounding fibro-osseous shea... more Flexor tendon repair in zone II is complicated by adhesions to the surrounding fibro-osseous sheath. Lactate is an early mediator of wound healing known to play an important role in stimulation of collagen production after cellular injury. Little attention has been paid to the role of lactate in flexor tendon wound healing. In this study tendon and tendon sheath were excised from rabbit forepaws. We examined proliferation of tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes; collagen production by each of these 3 cell types; and effects of lactate on cell proliferation and collagen production. Three cell lines, tendon sheath, epitenon, and endotenon, were isolated and cultured. Tendon sheath fibroblasts showed the greatest proliferation. All 3 cell lines produced collagen I, II, and III. Lactate significantly increased collagen production by all 3 cell lines. We show that cells of the tendon sheath, epitenon, and endotenon produce collagen in vitro. Modulation of lactate levels may provide a means to modulate collagen production.
Plastic and Reconstructive Surgery, 2001
Keloids represent a dysregulated response to cutaneous wounding that results in an excessive depo... more Keloids represent a dysregulated response to cutaneous wounding that results in an excessive deposition of extra-cellular matrix, especially collagen. However, the molec-ular mechanisms regulating this pathologic collagen dep-osition still remain to be elucidated. A previous ...
Journal of Hand Surgery-american Volume, 2007
Purpose: Tissue-engineered tendon grafts will meet an important clinical need. To engineer tendon... more Purpose: Tissue-engineered tendon grafts will meet an important clinical need. To engineer tendons, we used acellularized allogeneic tendon as scaffold material. To determine the ideal cell type to seed the scaffolds, we studied in vitro characteristics of epitenon tenocytes, tendon sheath fibroblasts, bone marrow-derived mesenchymal stem cells (BMSCs), and adipoderived mesenchymal stem cells (ASCs). Subsequently, we implanted reseeded acellularized tendons in vivo as flexor tendon grafts. Methods: Tenocytes, sheath fibroblasts, BMSCs, and ASCs were obtained from adult rabbits. For all cell lines, collagen 1, 2, and 3 immunocytochemistry was performed, and proliferation was assessed by hemacytometry and senescence by -galactosidase staining. Flexor tendons were acellularized after harvest. Tendons were assessed by histology after in vitro reseeding with each of the cell types after 1, 4, and 8 weeks. Finally, reseeded tendons and controls were implanted in a flexor profundus tendon defect. After 6 weeks, the reseeded tendons were harvested and assessed by histology. Statistical analysis for cell proliferation was performed using analysis of variance and t-tests with Bonferroni correction. Results: All cell types had similar collagen expression. Cell proliferation was higher in ASCs in late passage compared with early passage and in ASCs compared with epitenon tenocytes at late passage. The other cell types were similar in growth characteristics. No senescence was detected. In vitro assessment of reseeded constructs showed the presence of cells on the construct surface. In vivo assessment after implantation showed viable cells seen within the tendon architecture in all cell types.
Prostaglandins Leukotrienes and Essential Fatty Acids, 2001
Cytosolic phospholipase A 2 (cPLA 2 ) is believed to involve the regulation of essential cellular... more Cytosolic phospholipase A 2 (cPLA 2 ) is believed to involve the regulation of essential cellular processes. Like other cell types, epidermal cPLA 2 may participate in various metabolic processes including eicosanoid generation. In this investigation, we demonstrated the presence of cPLA 2 in guinea pig epidermis.The epidermal cPLA 2 is Ca 2+ -dependent, active at micromolar concentration of Ca 2+ and resistant to disulfide-reducing agents. Furthermore, it is inhibited by methyl arachidonyl fluorophosphonate (MAFP), a selective inhibitor of cPLA 2 , while12-epi-scalardial (a sPLA 2 inhibitor) did not cause inhibition. A test of several flavonoids revealed that quercetin (flavonol) weakly inhibited cPLA 2 , while flavanone had negligible inhibitory activity. In contrast, amentoflavone and ginkgetin (biflavones) markedly inhibited cPLA 2 activity in the epidermis. These results underscore that different flavonoids do vary in their capability to exert differential effects on arachidonate metabolism in the skin via modulation of epidermal cPLA 2 activity.
Journal of Hand Surgery-american Volume, 2004
Purpose: Postoperative adhesions frequently compromise the success of flexor tendon repair. Manip... more Purpose: Postoperative adhesions frequently compromise the success of flexor tendon repair. Manipulation of growth factors responsible for scar formation may be a method of decreasing adhesion formation. Transforming growth factor beta (TGF-) is a key cytokine in the pathogenesis of tissue fibrosis. The purpose of this study was to examine the effectiveness of TGF- neutralizing antibody in blocking TGF--induced collagen I production in rabbit flexor tendons in vitro. Methods: Sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were obtained from rabbit flexor tendons. Each cell culture was supplemented with 1 ng/mL of TGF- along with increasing doses of TGF- neutralizing antibody (0.1-2.0 g/mL). Collagen I production was measured by enzyme-linked immunoabsorbent assay and TGF- bioactivity was measured by the luciferase assay. Results were compared with TGF- alone and unsupplemented controls. Results: The addition of neutralizing antibody significantly reduced TGF--induced collagen I production in a dose-dependent manner in all 3 cell cultures. TGF- bioactivity was also reduced by its neutralizing antibody. Conclusions: This study shows that TGF- inhibition through its neutralizing antibody was effective in cultured flexor tendon cells. The results encourage further experiments that use such agents to modulate flexor tendon wound healing in in vivo models in the hope of eventually blocking the effect of TGF- on flexor tendons clinically. (J Hand Surg 2004;29A:230 -235.
Journal of Hand Surgery-american Volume, 2002
Flexor tendon healing is complicated by adhesions to the surrounding sheath. Transforming growth ... more Flexor tendon healing is complicated by adhesions to the surrounding sheath. Transforming growth factor beta (TGF-beta) is a cytokine with numerous activities related to wound healing. We examined the effects of TGF-beta-1, -2 and -3 on tendon cell proliferation and collagen production. Three separate cell lines--sheath fibroblasts, epitenon and endotenon tenocytes--were isolated from rabbit flexor tendons and cultured separately. Cell culture media was supplemented with 1 or 5 ng/mL of TGF-beta-1, -2, or -3. Cell number and collagen I and III production were measured and compared with unsupplemented control cultures. The addition of TGF-beta to cell culture media resulted in a decrease in cell number in all 3 lines that did not reach statistical significance. There was a significant increase (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;.05) in collagen I and III production with the addition of all 3 TGF-beta isoforms. Modulation of TGF-beta production may provide a mechanism to modulate adhesion formation clinically.
Tissue Engineering, 2006
A significant problem in flexor tendon repair is the lack of suitable graft material for reconstr... more A significant problem in flexor tendon repair is the lack of suitable graft material for reconstruction. The ex vivo production of flexor tendon graft constructs requires the expansion of primary cells. Growth factors, such as platelet-derived growth factor-BB (PDGF-BB), insulin-like growth factor-1 (IGF-1), and basic fibroblast growth factor (bFGF), are known to promote tendon healing and tendon cell proliferation. The purpose of these experiments was to optimize tenocyte proliferation in 3 tendon cell populations using growth factor supplementation. Cells of the synovial sheath, epitenon, and endotenon were isolated from rabbit flexor digitorum profundus tendons and maintained in culture. Cell cultures were supplemented with IGF-1, PDGF-BB, and bFGF alone and in combination. The conditions used for individual growth factor supplementation were IGF-1 (10, 50, and 100 ng/mL), PDGF-BB (1, 10, and 50 ng /mL), and bFGF (0.5, 1, and 5 ng/mL). The conditions used for combinations of growth factors were IGF-1 + PDGF-BB (50 + 10 and 100 + 50 ng /mL, respectively) and IGF-1 + PDGF-BB + bFGF (50 + 10 + 1; 50 + 10 + 5; 100 + 50 + 1; and 100 + 50 + 5 ng/mL, respectively). For all 3 tendon cell populations, proliferation at 72 h was greater in the presence of individual growth factors as compared to controls. With PDGF-BB (50 ng/mL) supplementation, mean absorbance values increased 97% (0.57 to 1.13) in S cells, 37% (0.51 to 0.70) in E cells, and 33% (0.33 to 0.44) in T cells ( p < 0.001). In addition, a synergistic effect was observed. The combination of growth factors resulted in greater proliferation as compared to maximal doses of individual growth factors. In cultures supplemented with IGF-1 (100 ng /mL) + PDGF-BB (50 ng/mL), mean absorbance increased 114% (0.57 to 1.22) in S cells, 63% (0.51 to 0.831) in E cells, and 47% (0.33 to 0.48) in T cells ( p < 0.001). IGF-1 (100 ng /mL) + PDGF-BB (50 ng/mL) + bFGF (5 ng/mL) resulted in the greatest amount of cell proliferation for all 3 tendon cell populations. The mean absorbances increased 251% in S cells, 98% in E cells, and 106% in T cells ( p < 0.001). In summary, IGF-1, PDGF-BB, and bFGF can be used in combination to maximize tenocyte proliferation. Synergism among growth factors may provide a means to facilitate tendon engineering.
Journal of Hand Surgery-american Volume, 2006
Purpose: Dupuytren's disease (DD) is characterized by fibroblastic proliferation of the palmar fa... more Purpose: Dupuytren's disease (DD) is characterized by fibroblastic proliferation of the palmar fascia, often leading to flexion contracture in the hand. Although there is a strong genetic component the genome-wide expression of novel genes is not known. The purpose of this study was to use DNA microarray technology to identify upregulated genes in DD. Methods: Human tissue samples were harvested from 3 patient sources: DD cord tissue (n ϭ 20), normal-appearing adjacent control fascia (n ϭ 15), and palmar fascia from patients having carpal tunnel release (n ϭ 15). DNA microarray analysis was performed on amplified sample RNA. Novel genes were compared with known gene functions. A candidate gene of interest was studied further by using immunohistochemistry on DD tissue samples and controls. Results: Several novel genes not described previously in the study of DD were upregulated significantly, including MafB, collagen type V, ␣-2 (COL5A2), collagen type VIII, ␣-1 (COL8A1), contactin I (CNTN1), and leucine-rich repeat containing 17 (LRRC17). These upregulated genes were compared with their known gene-expression profiles in other tissues and their purported functions. MafB was found to be of particular interest because of its prominent role in tissue development and cellular differentiation. MafB immunohistochemistry showed positive staining in 50% of the DD specimens but complete absence of MafB in all control tissues (adjacent control fascia, carpal tunnel fascia). Co-localization experiments with MafB and ␣-smooth muscle actin showed staining properties in similar regions but these 2 proteins were not confined solely to the same cells. Conclusions: Microarray analysis of DD tissue has identified significant upregulated gene expression of MafB. MafB protein also is found in Dupuytren's cords but not in control fascia. Co-localization data suggest that the association of MafB with DD is not related exclusively to myofibroblast proliferation. Because of its role in fibroblastic transformation in other models MafB and its relationship to the pathogenesis of DD deserves further study. (J Hand Surg 2006;31A: 211-218.
Journal of Hand Surgery-american Volume, 2001
Flexor tendon repair in zone II is complicated by adhesions to the surrounding fibro-osseous shea... more Flexor tendon repair in zone II is complicated by adhesions to the surrounding fibro-osseous sheath. Lactate is an early mediator of wound healing known to play an important role in stimulation of collagen production after cellular injury. Little attention has been paid to the role of lactate in flexor tendon wound healing. In this study tendon and tendon sheath were excised from rabbit forepaws. We examined proliferation of tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes; collagen production by each of these 3 cell types; and effects of lactate on cell proliferation and collagen production. Three cell lines, tendon sheath, epitenon, and endotenon, were isolated and cultured. Tendon sheath fibroblasts showed the greatest proliferation. All 3 cell lines produced collagen I, II, and III. Lactate significantly increased collagen production by all 3 cell lines. We show that cells of the tendon sheath, epitenon, and endotenon produce collagen in vitro. Modulation of lactate levels may provide a means to modulate collagen production.
Plastic and Reconstructive Surgery, 2001
Keloids represent a dysregulated response to cutaneous wounding that results in an excessive depo... more Keloids represent a dysregulated response to cutaneous wounding that results in an excessive deposition of extra-cellular matrix, especially collagen. However, the molec-ular mechanisms regulating this pathologic collagen dep-osition still remain to be elucidated. A previous ...
Journal of Hand Surgery-american Volume, 2007
Purpose: Tissue-engineered tendon grafts will meet an important clinical need. To engineer tendon... more Purpose: Tissue-engineered tendon grafts will meet an important clinical need. To engineer tendons, we used acellularized allogeneic tendon as scaffold material. To determine the ideal cell type to seed the scaffolds, we studied in vitro characteristics of epitenon tenocytes, tendon sheath fibroblasts, bone marrow-derived mesenchymal stem cells (BMSCs), and adipoderived mesenchymal stem cells (ASCs). Subsequently, we implanted reseeded acellularized tendons in vivo as flexor tendon grafts. Methods: Tenocytes, sheath fibroblasts, BMSCs, and ASCs were obtained from adult rabbits. For all cell lines, collagen 1, 2, and 3 immunocytochemistry was performed, and proliferation was assessed by hemacytometry and senescence by -galactosidase staining. Flexor tendons were acellularized after harvest. Tendons were assessed by histology after in vitro reseeding with each of the cell types after 1, 4, and 8 weeks. Finally, reseeded tendons and controls were implanted in a flexor profundus tendon defect. After 6 weeks, the reseeded tendons were harvested and assessed by histology. Statistical analysis for cell proliferation was performed using analysis of variance and t-tests with Bonferroni correction. Results: All cell types had similar collagen expression. Cell proliferation was higher in ASCs in late passage compared with early passage and in ASCs compared with epitenon tenocytes at late passage. The other cell types were similar in growth characteristics. No senescence was detected. In vitro assessment of reseeded constructs showed the presence of cells on the construct surface. In vivo assessment after implantation showed viable cells seen within the tendon architecture in all cell types.
Prostaglandins Leukotrienes and Essential Fatty Acids, 2001
Cytosolic phospholipase A 2 (cPLA 2 ) is believed to involve the regulation of essential cellular... more Cytosolic phospholipase A 2 (cPLA 2 ) is believed to involve the regulation of essential cellular processes. Like other cell types, epidermal cPLA 2 may participate in various metabolic processes including eicosanoid generation. In this investigation, we demonstrated the presence of cPLA 2 in guinea pig epidermis.The epidermal cPLA 2 is Ca 2+ -dependent, active at micromolar concentration of Ca 2+ and resistant to disulfide-reducing agents. Furthermore, it is inhibited by methyl arachidonyl fluorophosphonate (MAFP), a selective inhibitor of cPLA 2 , while12-epi-scalardial (a sPLA 2 inhibitor) did not cause inhibition. A test of several flavonoids revealed that quercetin (flavonol) weakly inhibited cPLA 2 , while flavanone had negligible inhibitory activity. In contrast, amentoflavone and ginkgetin (biflavones) markedly inhibited cPLA 2 activity in the epidermis. These results underscore that different flavonoids do vary in their capability to exert differential effects on arachidonate metabolism in the skin via modulation of epidermal cPLA 2 activity.