- Email: [email protected]
Acceleration of wound healing induced by enriched collagen solutions
Preliminary ACCELERATION ENRICHED
OF
WOUND COLLAGEN Preliminary
SHMUEL
SHOSHAN,
PH.D.,
AND
OUR PREVIOUSLY REPORTED [lo] succesful attempts to elicit a cell growth-promoting effect of enriched collagen solutions (ECS) led us to investigate the possibility of applying this preparation on wounds inflicted on soft tissue in order to bring about an acceleration in the healing process.
MATERIALS
AND
METHODS
Bilateral l-cm. long incisions were made through the back muscle of young ether anesthetized guinea pigs. Tritium-labeled collagen (3H-proline, 5 uCi./g. body weight) was extracted with cold 1 N NaCl from guinea pig-skin, and further purified by the trichloracetic acid-ethanol method, as described by Gross [2]. A 0.2% solution of collagen in NaCl-Tris buffer (ionic strength, 0.4; pH, 7.6) containing 50% Medium 199 [6] was allowed to form a semisolid gel at 37°C. which was then applied on the wound on one side prior to suturing. Sterility of the preparation was ensured by ultrafiltration of the From the Corrective Tissue Research Laboratory, Hebrew University Alpha Omega Research and Postgraduate Center, Faculty of Dental Medicine, P-0. Box 1172, erusalem, Israel. Supporte I by Research Grant 645141 from the National Institute of Dental Research, U. S. Department of Health, Education, and Welfare, Public Health Service. Submitted for publication May 6, 1970.
Repod
HEALING INDUCED SOLUTIONS
BY
Report SHIFRA
FINKELSTEIN,
M.SC.
solution prior to thermal gelation. The wound at the control side was sutured without treatment. All surgical procedures were carried out under strictly aseptic conditions. Healing was followed histologically and autoradiographically 3, 6, 11, and 21 days after infliction of the wounds. Autoradiography was performed using the liquid emulsion technique (Ilford K5 Nuclear Research Emulsion). Exposure time was 30 days. The histologic sections were stained with Harris’ hematoxylin and eosin (H. E.). All sections from both the experimental and control sites were given a code by the technician, but unknown to the examiners until the end of examination. This blind method of examination of histologic material is routinely used in our laboratory to ensure objective evaluation of the results.
RESULTS All the animals recovered from the operation and started to gain weight after 48 hours. A striking difference between the incision site containing ECS and that without treatment was apparent as early as on the 3rd postoperative day. Representative photomicrographs from the treated and untreated sites are shown in Fig. l-10. On the 3rd day, swelling, hemorrhage, and inflammation were conspicuously reduced on the experimental 48s
JOURNAL
OF SURGICAL
RESEARCH
VOL.
10
NO.
side as compared with the untreated controls (Figs. 1 and 2). The implanted ECS surrounded the incision site and few cells were seen within the implant (Fig. 3). Mature fibrocytes were present within the labeled collagen implant, and newly deposited fibers of nonlabeled collagen adjacent to the cells could be seen already on the 6th day after wounding (Fig. 4). At the control side, there was evidence of both hemorrhage and necrotic tissue debris at that time (Fig. 5). On the 11th day after wounding, when new collagen production in scar tissue is known to reach the maximum [S], a difference in the healing stage between the two incision sites was still obvious microscopically: while a “clean” scar with many newly formed, nonlabeled collagen fibers were seen in the ECStreated wounds (Figs. 6 and 7), the control wounds still had some necrotic foci sur-
LO, OCTOBER 1970
rounded by inflammatory cells (Fig. 8). At the end of the experiment, 3 weeks after wounding, the implanted ECS had a very dense population of fibrocytes and an almost entirely newly formed collagenous matrix, evident by the absence of silver grains following autoradiographic exposure and development (Fig. 9). The control wounds were also completely healed at that time, but microscopically, a few foci of degenerated muscle fibers and also small lymphocytes were still present.
DISCUSSION
The exact mechanism of the life maintaining repair processes, and how they are affected by intrinsic and/or extrinsic factors are as yet far from being understood. As to the
Fig. 1. Photomicrograph of wound area on the control side. Three days after wounding. Note necrosis, hemmorrhage, and swelling. H.E. X 105. Fig. 2. Photomicrograph of wound area on the ECS-treated side 3 days after wounding. On the left, the collagenous implant with marginal infiltration of cells. Note the relative “clean” wound area, with a strikingly lesser degree of both inflammation and hemorrhage as compared with the controls in Fig. 1. H.E. X 260. 486
SH0SHA.U
ASD
FINKELSTEIN:
ACCELERATIOX
OF WOUSD
HE.kLISG
Fig. 3. Microautoradiograph of ECS implant 3 days after wounding. A severed muscle fiber is seen at the lower part of the picture. Note proliferation of cells into the implant. H.E. X 650. Fig. 4. Microautoradiograph of ECS-treated wound area 6 days after wounding to show mature fibrocytes and newly synthetized nonlabeled collagen fibers within the implant (arrows). H.E. X 650.
mechanism by which our ECS preparation acts, it seems valid, though, to speculate that both hemmorrhage and edema are reduced via the known effect of collagen on platelet aggregation and blood clotting (4, 7, 12), while the included cell nutrients, kept in situ by the collagen network, may contribute to fibroblast proliferation and, consequently, to new fiber formation. Several reports on the beneficial effect of soluble collagen on the healing of dermal incision wounds were published during past years [3]. It has been shown that the tensile strength of scars was considerably increased following several injections of collagen solutions into the wound area [ll]. Our results were obtained with enriched collagen solutions which were thermally gelled before use. This indicates that a single direct application of the ECS preparation elicits a beneficial effect even earlier (on the 3rd postoperative
day already). The disappearance of tritiumlabeled collagen, beginning with the 6th postoperative day, indicates metabolic activity at the implantation site, which ultimately brings about complete removal of the implanted ECS and its replacement by host-produced collagen. This fact, as well as the low immunogenicity of collagen [ 1, 31, points to the possible application of our ECS preparation in clinical practice, thus eliminating the need to use preparations containing chemicals or hormones [5, 91 which may often be clinicall>contraindicated. It seems that our preparatiou fulfils the two main pharmacologic criteria required from a drug, namely, safety and efficacy. Further studies on the effect of enriched collagen preparations on healing of fractures and burn wounds and their clinical applicability are currently in progress in our laboratory. 487
JOURNAL
Fig. 5. necr
OF SURGICAL
RESEARCH
VOL.
Photomicrograph of a control tissue debris and inflammation.
10
NO.
10,
OCTOBER
1970
wound section 6 days after wounding H.E. X 105.
to
presence of both
SHOSHAN
AND
FINKELSTEIN:
ACCELERATION
OF WOUND
HEALING
Fig. 6.
Photomicrograph of ECS-treated wound area 11 days after wounding. Note the fibrc xyterich ECS implant, the clean scar, and the absence of tissue debris and inflammation. H.E. X 105. of ECS implant 11 days after wounding to show cell growth and Inewly Fig. 7. Microautoradiograph fonr Led collagen fibers. H.E. X 650.
489
JOURNAL
Fig. 8.
OF
SURGICAL
RESEARCH
Photomicrograph of also the presence of necrosis Fig. 9. Photomicrograph of wou nd consisting of cellular fiber s. H.E. X 105.
490
VOL.
10
NO.
10,
OCTOBER
1970
a control wound 11 days after wounding. Note the organized scar but and inflammation at the lower right corner. H.E. X 105. ECS-treated wound 21 days after wounding to show a complete he raled fibrous tissue filling the gap between the cut edges of the injured ml 1scle
SHOSHAN
AND
FlSKELSTEIN:
ACCELERATION
OF
WOUND
HF,ALIXG
Fig. 10. Microautoradiograph of ECS-treated wound 21 days after wounding to show an almost complete absence of labeled collagen at the implantation site and a dense population of new collagenproducing fibrocytes. H.E. X 650. REFERENCES H. C., and Gross, J. Thermal recon1. Grillo, stitution of collagen from solution and the response to its heterologous implantation. J. SuTg. Res. 2:69, 1962. on formation of collagen. 2. Gross, J. Studies I Properties and fractionation of neutral salt extracts of normal guinea pig connective tissue. J. Exp. Med. 107:247, 1958. H. J,, and Struck, H. 3. Hernandez-Richter, Neuere Probleme der Wundheilung. Int. Surg. 48:385, 1967. C. M. Nouvelles J., and LapiBre, 4. Hughes, recherches sur l’accollement des plasquettes aw fibres de collagkn. Thromb. Diath. haemorrh. 11:327, 1964. W. R. Enhanced healing of skin 5. Klemm, wounds in dogs with systemically and locally administered drugs. Experientia 23~55, 1967. 6. Morgan, J. F., Morten, N. J., and Parker, R. C. Nutrition of animal cells in tissue culture. I. Initial studies on a synthetic medium. PTOC. Sot. Exp. Biol. Med. 73:1, 1950.
7.
Niewarowski, S, and Bankowski, E. Interaction of the Hageman Factor (Factor XII) with insoluble collagen. Bull. Acad. Pal. Sci. 12:137, 1969.
8. Peacock, E. E., Jr. Production and polymerization of collagen in healing wounds in rats: some rate-regulating factors. Ann. Surg. 155:251, 1962. 9. Rosenthal, S. P. Acceleration of primary wound healing by insulin. Arch. Surg. 96:53, 1968. 10.
Shoshan, S., and Finkelstein, S. Cell growth promoting effect of enriched collagen solutions thermally gelled in vivo. Israel J. Med. Sci. 3: 755, 1967.
11.
Struck, H. Experimentelle Untersuchungen zur Verbesserung der Wundheilung. In “Die Entzuendung-Grundlagen und Pharmakologische Beeinflussung.” Muenchen, Berlin, Wien: Urban and Schwarzenberg, 1966. PP. 3-11.
12. Zucker, M. B., and Borrelli, J. Platelet clumping produced by connective tissue suspensions and by collagen. Proc. Sot. Exp. Biol. Med. 109: 779, 1962.
491
OF
WOUND COLLAGEN Preliminary
SHMUEL
SHOSHAN,
PH.D.,
AND
OUR PREVIOUSLY REPORTED [lo] succesful attempts to elicit a cell growth-promoting effect of enriched collagen solutions (ECS) led us to investigate the possibility of applying this preparation on wounds inflicted on soft tissue in order to bring about an acceleration in the healing process.
MATERIALS
AND
METHODS
Bilateral l-cm. long incisions were made through the back muscle of young ether anesthetized guinea pigs. Tritium-labeled collagen (3H-proline, 5 uCi./g. body weight) was extracted with cold 1 N NaCl from guinea pig-skin, and further purified by the trichloracetic acid-ethanol method, as described by Gross [2]. A 0.2% solution of collagen in NaCl-Tris buffer (ionic strength, 0.4; pH, 7.6) containing 50% Medium 199 [6] was allowed to form a semisolid gel at 37°C. which was then applied on the wound on one side prior to suturing. Sterility of the preparation was ensured by ultrafiltration of the From the Corrective Tissue Research Laboratory, Hebrew University Alpha Omega Research and Postgraduate Center, Faculty of Dental Medicine, P-0. Box 1172, erusalem, Israel. Supporte I by Research Grant 645141 from the National Institute of Dental Research, U. S. Department of Health, Education, and Welfare, Public Health Service. Submitted for publication May 6, 1970.
Repod
HEALING INDUCED SOLUTIONS
BY
Report SHIFRA
FINKELSTEIN,
M.SC.
solution prior to thermal gelation. The wound at the control side was sutured without treatment. All surgical procedures were carried out under strictly aseptic conditions. Healing was followed histologically and autoradiographically 3, 6, 11, and 21 days after infliction of the wounds. Autoradiography was performed using the liquid emulsion technique (Ilford K5 Nuclear Research Emulsion). Exposure time was 30 days. The histologic sections were stained with Harris’ hematoxylin and eosin (H. E.). All sections from both the experimental and control sites were given a code by the technician, but unknown to the examiners until the end of examination. This blind method of examination of histologic material is routinely used in our laboratory to ensure objective evaluation of the results.
RESULTS All the animals recovered from the operation and started to gain weight after 48 hours. A striking difference between the incision site containing ECS and that without treatment was apparent as early as on the 3rd postoperative day. Representative photomicrographs from the treated and untreated sites are shown in Fig. l-10. On the 3rd day, swelling, hemorrhage, and inflammation were conspicuously reduced on the experimental 48s
JOURNAL
OF SURGICAL
RESEARCH
VOL.
10
NO.
side as compared with the untreated controls (Figs. 1 and 2). The implanted ECS surrounded the incision site and few cells were seen within the implant (Fig. 3). Mature fibrocytes were present within the labeled collagen implant, and newly deposited fibers of nonlabeled collagen adjacent to the cells could be seen already on the 6th day after wounding (Fig. 4). At the control side, there was evidence of both hemorrhage and necrotic tissue debris at that time (Fig. 5). On the 11th day after wounding, when new collagen production in scar tissue is known to reach the maximum [S], a difference in the healing stage between the two incision sites was still obvious microscopically: while a “clean” scar with many newly formed, nonlabeled collagen fibers were seen in the ECStreated wounds (Figs. 6 and 7), the control wounds still had some necrotic foci sur-
LO, OCTOBER 1970
rounded by inflammatory cells (Fig. 8). At the end of the experiment, 3 weeks after wounding, the implanted ECS had a very dense population of fibrocytes and an almost entirely newly formed collagenous matrix, evident by the absence of silver grains following autoradiographic exposure and development (Fig. 9). The control wounds were also completely healed at that time, but microscopically, a few foci of degenerated muscle fibers and also small lymphocytes were still present.
DISCUSSION
The exact mechanism of the life maintaining repair processes, and how they are affected by intrinsic and/or extrinsic factors are as yet far from being understood. As to the
Fig. 1. Photomicrograph of wound area on the control side. Three days after wounding. Note necrosis, hemmorrhage, and swelling. H.E. X 105. Fig. 2. Photomicrograph of wound area on the ECS-treated side 3 days after wounding. On the left, the collagenous implant with marginal infiltration of cells. Note the relative “clean” wound area, with a strikingly lesser degree of both inflammation and hemorrhage as compared with the controls in Fig. 1. H.E. X 260. 486
SH0SHA.U
ASD
FINKELSTEIN:
ACCELERATIOX
OF WOUSD
HE.kLISG
Fig. 3. Microautoradiograph of ECS implant 3 days after wounding. A severed muscle fiber is seen at the lower part of the picture. Note proliferation of cells into the implant. H.E. X 650. Fig. 4. Microautoradiograph of ECS-treated wound area 6 days after wounding to show mature fibrocytes and newly synthetized nonlabeled collagen fibers within the implant (arrows). H.E. X 650.
mechanism by which our ECS preparation acts, it seems valid, though, to speculate that both hemmorrhage and edema are reduced via the known effect of collagen on platelet aggregation and blood clotting (4, 7, 12), while the included cell nutrients, kept in situ by the collagen network, may contribute to fibroblast proliferation and, consequently, to new fiber formation. Several reports on the beneficial effect of soluble collagen on the healing of dermal incision wounds were published during past years [3]. It has been shown that the tensile strength of scars was considerably increased following several injections of collagen solutions into the wound area [ll]. Our results were obtained with enriched collagen solutions which were thermally gelled before use. This indicates that a single direct application of the ECS preparation elicits a beneficial effect even earlier (on the 3rd postoperative
day already). The disappearance of tritiumlabeled collagen, beginning with the 6th postoperative day, indicates metabolic activity at the implantation site, which ultimately brings about complete removal of the implanted ECS and its replacement by host-produced collagen. This fact, as well as the low immunogenicity of collagen [ 1, 31, points to the possible application of our ECS preparation in clinical practice, thus eliminating the need to use preparations containing chemicals or hormones [5, 91 which may often be clinicall>contraindicated. It seems that our preparatiou fulfils the two main pharmacologic criteria required from a drug, namely, safety and efficacy. Further studies on the effect of enriched collagen preparations on healing of fractures and burn wounds and their clinical applicability are currently in progress in our laboratory. 487
JOURNAL
Fig. 5. necr
OF SURGICAL
RESEARCH
VOL.
Photomicrograph of a control tissue debris and inflammation.
10
NO.
10,
OCTOBER
1970
wound section 6 days after wounding H.E. X 105.
to
presence of both
SHOSHAN
AND
FINKELSTEIN:
ACCELERATION
OF WOUND
HEALING
Fig. 6.
Photomicrograph of ECS-treated wound area 11 days after wounding. Note the fibrc xyterich ECS implant, the clean scar, and the absence of tissue debris and inflammation. H.E. X 105. of ECS implant 11 days after wounding to show cell growth and Inewly Fig. 7. Microautoradiograph fonr Led collagen fibers. H.E. X 650.
489
JOURNAL
Fig. 8.
OF
SURGICAL
RESEARCH
Photomicrograph of also the presence of necrosis Fig. 9. Photomicrograph of wou nd consisting of cellular fiber s. H.E. X 105.
490
VOL.
10
NO.
10,
OCTOBER
1970
a control wound 11 days after wounding. Note the organized scar but and inflammation at the lower right corner. H.E. X 105. ECS-treated wound 21 days after wounding to show a complete he raled fibrous tissue filling the gap between the cut edges of the injured ml 1scle
SHOSHAN
AND
FlSKELSTEIN:
ACCELERATION
OF
WOUND
HF,ALIXG
Fig. 10. Microautoradiograph of ECS-treated wound 21 days after wounding to show an almost complete absence of labeled collagen at the implantation site and a dense population of new collagenproducing fibrocytes. H.E. X 650. REFERENCES H. C., and Gross, J. Thermal recon1. Grillo, stitution of collagen from solution and the response to its heterologous implantation. J. SuTg. Res. 2:69, 1962. on formation of collagen. 2. Gross, J. Studies I Properties and fractionation of neutral salt extracts of normal guinea pig connective tissue. J. Exp. Med. 107:247, 1958. H. J,, and Struck, H. 3. Hernandez-Richter, Neuere Probleme der Wundheilung. Int. Surg. 48:385, 1967. C. M. Nouvelles J., and LapiBre, 4. Hughes, recherches sur l’accollement des plasquettes aw fibres de collagkn. Thromb. Diath. haemorrh. 11:327, 1964. W. R. Enhanced healing of skin 5. Klemm, wounds in dogs with systemically and locally administered drugs. Experientia 23~55, 1967. 6. Morgan, J. F., Morten, N. J., and Parker, R. C. Nutrition of animal cells in tissue culture. I. Initial studies on a synthetic medium. PTOC. Sot. Exp. Biol. Med. 73:1, 1950.
7.
Niewarowski, S, and Bankowski, E. Interaction of the Hageman Factor (Factor XII) with insoluble collagen. Bull. Acad. Pal. Sci. 12:137, 1969.
8. Peacock, E. E., Jr. Production and polymerization of collagen in healing wounds in rats: some rate-regulating factors. Ann. Surg. 155:251, 1962. 9. Rosenthal, S. P. Acceleration of primary wound healing by insulin. Arch. Surg. 96:53, 1968. 10.
Shoshan, S., and Finkelstein, S. Cell growth promoting effect of enriched collagen solutions thermally gelled in vivo. Israel J. Med. Sci. 3: 755, 1967.
11.
Struck, H. Experimentelle Untersuchungen zur Verbesserung der Wundheilung. In “Die Entzuendung-Grundlagen und Pharmakologische Beeinflussung.” Muenchen, Berlin, Wien: Urban and Schwarzenberg, 1966. PP. 3-11.
12. Zucker, M. B., and Borrelli, J. Platelet clumping produced by connective tissue suspensions and by collagen. Proc. Sot. Exp. Biol. Med. 109: 779, 1962.
491