Beneficial wound healing applications of calreticulin and other hyaluronan-associated proteins

Hyaluronan associated proteins, in particular calreticulin, promote the accelerated and relatively scarless healing of wounds. Methods for treating wounds using such proteins, and pharmaceutical compositions comprising such proteins, are provided.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention in the field of medicine relates to novel 
compositions and methods for the treatment of wounds and for the promotion 
of more rapid wound healing with diminished scarring. 
2. Description of the Background Art 
Despite significant progress in reconstructive surgical techniques, 
scarring can be an important obstacle in regaining normal function and 
appearance of healed skin. This is particularly true when pathologic 
scarring such as keloids or hypertrophic scars of the hands or face causes 
functional disability or physical deformity. In the severest 
circumstances, such scarring may precipitate psychosocial distress and a 
life of economic deprivation. 
Healing wounded tissue is among the most essential, dramatic and visible 
jobs performed by the body. Significant progress has recently been made in 
understanding the sequence of events occurring when traumatized tissue 
heals. Several dozen different growth factors, or cytokines, have been 
identified that participate in healing. These growth factors signal the 
blood to coagulate and plug the gap, they attract immune cells to fight 
infiltrating microorganisms, and ultimately promote neighboring skin cells 
to cover the wound. If the wound is sufficiently large, these factors 
stimulate production of new skin, new blood vessels, new supporting 
connective tissue and even new bone. 
Adult Wound Healing 
Adult wound healing in response to injury results in restoration of tissue 
continuity (Adzick N. S. et al. (eds), FETAL WOUND HEALING, Elsevier, New 
York 1992, Chapters 1-3, 12, 13 and references cited therein). While some 
amphibians heal by regeneration, adult mammalian tissue repair involves a 
complex series of biochemical events that ultimately ends in scar 
formation. The events occurring during wound repair resemble the process 
of development, including synthesis, degradation and resynthesis of the 
extracellular matrix (ECM) (Smith L. T. et al. (1982) J Invest Dermatol 
79:935-1045; Blanck C. E. et al. (1987) J Cell Biol 105:139(A)). The ECM 
contains several macromolecules, including collagen, fibronectin, fibrin, 
proteoglycans, and elastin (Cohen J. K. et al. (1983) BIOCHEMISTRY AND 
PHYSIOLOGY OF THE SKIN. New York: Oxford University Press, pp 462-470, 
1983; Alvarez O. M., In: CONNECTIVE TISSUE DISEASE: MOLECULAR PATHOLOGY OF 
THE EXTRACELLULAR MATRIX, Uitto J. et al., eds, New York: Marcell Decker, 
pp. 367-384, 1986; Murphy-Ullrich J. E. et al., supra, at pp. 455-473). 
When the injury involves the dermis, repair also entails the removal of 
cellular debris (Grinnel F. et al. (1981) J Invest Dermatol 76:181-189) 
and the laying down of a new ECM over which epidermal continuity can be 
reestablished. This process of repair and dermal matrix reorganization is 
manifested as scar formation and maturation. 
Microscopically, the scar can be identified by its abnormal organization of 
cellular and matrix elements when compared to surrounding uninjured skin. 
Grossly, normal scars progress towards stability and maturity. An immature 
scar is raised, red, and firm, whereas a mature scar is flat, white, and 
soft. However, not all healing follows this pattern and can result in 
abnormal scars, such as hypertrophic or keloid scars. Both of these types 
of scars can be differentiated clinically and histomorphologically from 
normal scar, but this invariably involves repeated observation over a 
period of time, as hypertrophic scars in particular can progress to the 
maturity of a normal scar albeit over a much longer time course. 
Adult wound repair includes the stages of hemostasis, inflammation, 
proliferation, and remodeling. Hemostasis includes vasoconstriction, 
platelet aggregation and degranulation, blood clotting, and fibrin 
formation. Inflammation represents a cellular cascade beginning with 
polymorphonuclear leukocytes (PMNs) followed by macrophages and 
lymphocytes. This stage also provides host defenses against bacterial 
infection and contributes numerous growth factors, cytokines, and 
extracellular matrix (ECM) components. The wound macrophage is the crucial 
inflammatory effector cell that coordinates adult wound repair (Knighton 
D. R. et al. (1989) Prog Clin Biol Res 299:217-226). 
The proliferative stage involves multiplication of fibroblasts and 
endothelial and epithelial cells. The initial proteoglycan-rich fibrin 
matrix is replaced by collagen. In the final remodeling stage, collagen is 
cross-linked to form a mature scar. In abnormal wound healing conditions 
such as keloids, hypertrophic scars, strictures, and intraabdominal 
adhesions, the final result of wound repair creates a cosmetic or 
functional problem. 
Based on the fact that scar formation and maturation involves a complex 
interaction of dermal and epidermal cells with the ECM, an artificial ECM 
model has been used to guide the laying down of a new ECM which results in 
less scarring (Yannis I. V. et al. (1989) Proc Natl Acad Sci U.S.A. 
86:933-937). Tension can influence the orientation of organizing collagen, 
based both on clinical observations and in vitro studies of contracting 
collagen matrices (Burd D. et al. (1989) Proc Amer Burns Assoc, p. 54). 
Growth Factors and Wound Healing 
Manipulation of the wound healing environment by the application of 
extrinsic growth factors such as fibroblast growth factor (FGF) and 
transforming growth factor-.beta. (TGF.beta.) (Mustoe T. A. et al. (1987) 
Science 237:1333-1336; Seyedin S. M. et al. (1986) J Biol Chem 
261:5693-5695) can influence the early stages of scar formation. The term 
"TGF.beta." represents a family of 25 kDa dimeric proteins that influence 
important cell-cell and cell-matrix interactions during embryogenesis, 
immune responses, and tissue repair. During tissue repair, TGF.beta. 
modulates the inflammatory response as a potent chemoattractant for 
fibroblasts, macrophages, neutrophils and T lymphocytes. TGF.beta.1 
promotes ECM accumulation by increasing the transcription of genes for 
collagen, fibronectin and glycosaminoglycans and by inhibiting the 
breakdown of these macromolecules (as described herein). TGF.beta. can 
also up-regulate cell surface expression of the integrins that act as 
receptors for fibronectin, collagen, laminin, and vitronectin thereby 
influencing cell adhesion and migration. TGF.beta. enhances the epithelial 
covering of exposed dermis and increases tensile strength in incisional 
wounds. 
Three mammalian isoforms of TGF.beta. are known which exhibit an 80% amino 
acid sequence homology. Until recently, the TGF.beta. isoforms were 
thought to be functionally identical, although more recent demonstration 
of different in vivo effects compared to in vitro activity, and knowledge 
of the distinction between the three isoforms has prompted further 
analysis. Immunohistochemical analysis using anti-peptide antibodies 
specific for each TGF.beta. isoform has shown distinct expression patterns 
for each isoform in embryogenesis and carcinogenesis. Distinct promoters 
for the human TGF.beta.1, TGF.beta.2, and TGF.beta.3 genes provides a 
mechanism for the observed differential expression in selected tissues. 
This data coupled with the fact that the three isoforms are 98% conserved 
across species implies both specific function and complex gene regulation 
for each TGF.beta. isoform in vivo, reinforcing the notion that the three 
isoforms are not simply interchangeable (Seyedin et al., supra). During 
repair, specific roles for TGF.beta. isoforms are poorly understood. 
Fetal Wound Healing 
Human fetal surgery has been successfully performed to treat 
life-threatening fetal urinary tract obstruction and diaphragmatic hernias 
(Harrison M. R. et al. (1982) N Engl J Med 306:591-593; Harrison M. R. et 
al. (1987) J Pediatr Surg 22:556-558). Following the successful delivery 
of such babies, it has been observed that scarring or contracture around 
the decompressing hydronephrostomy tubes was absent. Numerous studies have 
shown that fetal wounds heal without scarring (Adzick N. S. et al. (1985) 
J Ped Surg 20:315-319; Siebert J. W. et al. (1990) Plast Reconstr Surg 
85:495-502). Immunohistochemical and biochemical studies (Longaker M. T. 
et al. (1990) J Ped Surg 25:63-69; Adzick et al., supra; Burd D. et al. 
(1990) Brit J Plast Surg 43:571-577) indicate that, as in adults, fetal 
skin wounds also possess a repair matrix which includes collagen. However 
in contrast to adult healing, the matrix is rapidly and efficiently 
organized to appear scarless. 
The present invention is intended to exploit knowledge gained from work on 
fetal wound healing and describe the sequencing of a putative fetal 
protein factor involved in collagen and matrix organization. 
Environmental Differences 
Numerous intrinsic and extrinsic differences between the fetus and the 
adult may drastically influence wound repair. Fetal skin wounds are 
continually bathed in warm, sterile amniotic fluid rich in growth factors 
that are crucial to fetal development (Azdick et al., supra). Amniotic 
fluid is also a rich source for ECM components such as hyaluronan (HA) and 
fibronectin. Amniotic fluid could modulate fetal skin wound repair simply 
by supplying HA and fibronectin directly onto fetal skin wounds and by 
providing growth factors to simulate fetal wound cells to make a unique 
wound matrix (Azdick et al., supra). 
To investigate the influence of the fetal environment on adult tissue 
repair, full-thickness sheep skin was transplanted onto the backs of 
60-day fetal lambs (term=145 days) (Azdick et al., supra), which at that 
age do not reject allogeneic skin grafts. The adult skin graft was thus 
bathed in amniotic fluid and perfused by fetal blood; 40 days later (at 
100 days gestation), incisional wounds were made on both the adult skin 
grafts and adjacent fetal skin, and immunohistochemical analysis was 
performed 7 and 14 days post-wounding. By 14 days the fetal wounds had 
healed without scarring, while the adult wound collagen pattern was in a 
typical scar pattern. Thus, neither the amniotic fluid environment nor 
perfusion by fetal blood prevented scar formation in the wounded adult 
skin graft. This suggested that the ability of fetal skin to heal without 
scar formation may be a function of the fetal cells and matrix with or 
without a fetal environmental influence. 
Intrinsic environmental differences include fetal tissue oxygenation, as 
the fetus depends on transplacental transport from the maternal 
circulation to meet its oxygen requirements. Because there is a large 
transplacental oxygen gradient between maternal arterial and umbilical 
venous blood, fetal arterial blood has a very low pO.sub.2 of 20 torr, 
which is lower than a maskless mountaineer on top of Mt. Everest (Azdick 
et al., supra). Fetal wound healing in the face of low fetal arterial 
pO.sub.2 seems paradoxical. The answer may lie in an inherent difference 
between the responsiveness of fetal and adult fibroblasts to differing 
levels of hypoxemia (Longaker M. T. et al. (1993) Plast Surg Res Council). 
Some of the properties of fetal skin wound healing may reflect the 
development of fetal skin. However, healing of fetal bone is also 
different from adult bone. Virtually no callus formation is present at any 
time during the healing of fetal lamb bone, and healed fracture sites are 
indistinguishable radiologically and histologically from uninjured bone. 
In addition, large bony defects in the fetus, which would be unhealable in 
infants or adults do close. Not all fetal tissues appear to share the 
remarkable regenerative qualities of fetal skin and bone. In in utero 
repair of previously surgically created fetal diaphragmatic hernias, the 
fetal intestine was always densely adherent to the diaphragmatic defect, 
but no scar was evident on the previously made thoracic skin incision. 
Clinical experience with human fetal surgery has shown extensive 
intraabdominal adhesions following fetal diaphragmatic hernia repair. 
Thus, fetal mesothelial wounds may heal differently from fetal skin 
wounds. In addition, amniotic fluid exposure may play an important role in 
the scarless healing of fetal skin wounds, but its effect on the healing 
of fetal mesothelial wounds has not been demonstrated. 
Fetal Inflammation 
Another intrinsic difference between the fetus and adult lies in the 
inflammatory and immune systems. Histologically, there are few, if any, 
PMNs in fetal wounds, and there may be a defect in immature PMN 
chemotactic ability. Fetal lamb wounds lack the typical inflammatory 
response seen in adult sheep (Longaker M. T. et al., 1990, supra). Because 
of the prominent role that inflammation plays in adult tissue repair, the 
minimal fetal inflammatory response to injury may play a pivotal role in 
the unique fetal repair process. Introduction of adult acute inflammatory 
cells into the fetus attracts fetal PMNs to the wound site, but an adult 
fibrotic type of healing response does not follow. These intriguing 
findings raise questions regarding what attracts fetal fibroblasts into 
the wounds, how this differs between fetus and adult, and whether 
characteristic inflammatory mediators of adult wound healing are absent in 
fetal wounds. 
The wound macrophage is the crucial inflammatory cell orchestrating adult 
wound healing (Knighton et al., supra). Neutrophils can be eliminated from 
wound repair without a defect in granulation tissue but macrophages 
cannot. Macrophages are essential regulatory cells that coordinate matrix 
debridement and turnover, and secrete mediators of inflammation, 
angiogenesis, and cell growth (Knighton et al., supra). Fetal rabbit 
wounds, though lacking in PMNs, have an abundance of macrophages (Adzick 
N. S. et al., 1985, supra). In addition to regulation through growth 
factor expression, wound macrophages are involved in matrix turnover 
through proteinase expression. Their secretion of metalloproteinases 
(e.g., collagenase) and proteinase inhibitors coordinates the degradation 
and remodeling of the wound ECM. The observation that fetal lamb 
incisional wounds appear histologically indistinguishable from unwounded 
skin within two weeks suggests that fetal wound matrix turnover and repair 
are rapid and efficient (Longaker M. T. et al., 1990, supra). 
Fetal Growth Factors 
In the fetus, wounds made before the mid-third trimester heal with a 
collagen repair matrix so organized as to appear scarless, but as in 
adults, growth factors can modulate the healing wound. 
Addition of TGF.beta. or PDGF converted a fetal injury response to an 
adult-like injury response (Krummel T. M. et al. (1988) J Ped Surg 
23:647-652). Administration of anti-TGF.beta. antibodies blocked the 
increased fibrosis in a wound treated with TGF.beta.1 (Shah Met al. (1992) 
Lancet 339:213-214). These results further implicate TGF.beta. in scar 
formation. In fetal mouse lip wounds that normally heal without scarring, 
the presence of TGF.beta.1 or .beta.2 isoforms could not be detected 
immunohistochemically with neutralizing antibodies (Whitby D. J. et al. 
(1991) Dev Biol 147:207-215). This is in stark contrast to neonatal and 
adult lip wounds which did immunostain for both isoforms. However, it has 
been shown that fetal wound fluid is abundant in TGF.beta. even during the 
period of scarless healing, although, interestingly, there is a change in 
the relative concentrations of isoforms as gestation progresses (Roberts 
A. B. et al. (1993) J Cell Biol Supplement 17E). 
Thus, the presence of growth factors in vivo in healing wounds demonstrated 
by immunohistochemical staining, neutralizing antibody techniques, and 
direct assay of wound chamber fluid, supports the concept that growth 
factors are important in modulating wound healing in the fetus as in the 
adult. 
Hyaluronan 
Hyaluronan (HA), formerly called hyaluronic acid or hyaluronate (Balaz E. 
A. et al. (1986) Biochem J. 233:903), is found in high concentration in 
ECM wherever tissue repair occurs after injury (Toole, B. P., In: Hay E. 
D., ed., CELL BIOLOGY OF THE EXTRACELLULARMATRIX. New York: Plenum Press; 
pp. 259-294, 1982). HA is a glycosaminoglycan (GAG) laid down early in the 
matrix of both fetal and adult wounds. Sustained deposition of HA is 
unique to fetal skin, where injury repair occurs with less scarring and 
more rapidly than adult injury repair. HA appears to provide an 
extracellular environment conducive to cell mobility and proliferation 
that may provide the matrix signal responsible for orchestrating healing 
by regeneration rather than by scarring in the fetus. The fetal wound 
matrix is rich in HA (Krummel T. M. et al., 1987, supra; De Palma R. L. et 
al. (1987) Surg Forum 38:626-628; De Palma R. L. et al. (1989) Matrix 
9:224-231)). By implanting PVA sponges into 24 day fetal rabbits or into 
adult rabbits, it was found that the GAG content of fetal sponges was 
significantly greater on day 2 through 6 when compared to adult sponges, 
and had 10 times the amount of GAG found in unwounded fetal skin. The 
major GAG component was HA, as determined by cellulose acetate 
electrophoresis followed by alcian blue staining (DePalma et al., 1989, 
supra; Longaker M. T. et al. (1989) Ann Surg 210:667-672). 
A role for HA in the scarless healing in the fetus is supported by studies 
in which topical application of HA tissue extracts modulated post-natal 
healing, and, for example, enhanced wound healing in rat tympanic membrane 
perforations (Hellstrom S. et al. (1987) Acta Otolaryngol 442 
(Suppl):7-24). HA facilitated wound healing in diabetic rats by promoting 
epithelial migration and differentiation (Abatangelo G. et al. (1983) J 
Surg Res 35:410-416). HA-treated wounds developed a greater early wound 
breaking strength compared to untreated controls, reportedly due to an 
early accumulation of oriented collagen fibers (Radelli E. et al. (1982) 
Int'l. Symp. Cutaneous Development, Aging and Repair, University of 
Padova, p. 42). However, attributing the wound healing effects exclusively 
to HA is difficult. It is important to remember that tissue-extracted HA, 
for example from rooster comb or human umbilical cord, is always 
"contaminated" with one or more proteins, including collagen (Swann D. A. 
et al. (1975) Ann Rheum Dis 34 (Suppl):98-100). 
The present inventors and their colleagues identified a heterogenous group 
of HA-protein complexes in normal skin and post-burn scar and confirmed 
the association of HA and collagen. Further, they found that HA extracted 
from normal skin, normal scar, and hypertrophic scar demonstrated 
qualitative and quantitative variation in other non-collagen associated 
proteins despite identical extraction and purification techniques (Burd D. 
A. R. et al. (1989) Matrix 9:322-327). 
SUMMARY OF THE INVENTION 
The present inventors have conceived of the use of hyaluronan associated 
proteins (HA-AP), in particular a protein appearing as a 62 kDa HA-AP, 
calreticulin, for promoting the scarless healing of wounds, such as 
surgical wounds or wounds incurred in accidental trauma. 
The present invention is therefore directed to a method of promoting the 
more rapid healing of a wound with diminished scar formation in a subject, 
comprising administering to the subject in need of such treatment an 
amount of a hyaluronan-associated protein, or a functional derivative 
thereof, effective in promoting scarless healing of wounds. 
Also provided is a method for modulating the expression of TGF.beta. 
isoforms in the healing tissue of a wound in a subject, such that 
TGF.beta.3 expression is enhanced and TGF.beta.1 expression and TGF.beta.2 
expression are inhibited, which method comprises administering to the 
subject an amount of a hyaluronan-associated protein, or a functional 
derivative thereof, effective in enhancing TGF.beta.3 and inhibiting 
TGF.beta.1 and TGF.beta.2. 
In the above methods, the hyaluronan-associated protein preferably has an 
apparent molecular weight of about 62 kDa upon SDS-PAGE under reducing 
conditions. Most preferably, the protein is calreticulin. 
The above methods may further comprise administering to the subject, in 
combination with the hyaluronan-associated protein or functional 
derivative, an effective amount of at least one other agent useful in 
promoting the healing of a wound. A preferred agent is an anti-bacterial 
agent, an anti-viral agent, an anti-fungal agent, a local anesthetic, an 
analgesic, and a growth factor. Preferred growth factors include 
transforming growth factor-.alpha., transforming growth factor-.beta., 
fibroblast growth factor-.alpha., fibroblast growth factor-.beta., 
epidermal growth factor, platelet-derived growth factor, endothelial 
cell-derived growth factor, insulin-like growth factors, and granulocyte 
colony-stimulating factor. 
In the above methods, the hyaluronan-associated protein, or calreticulin, 
or functional derivative, may be administered in a form associated with a 
solid or semisolid phase support material. 
The above methods are preferably used for treating a wound caused by 
physical or surgical trauma. 
The present invention is also directed to a pharmaceutical composition 
useful in the promotion of scarless wound healing, comprising: 
(a) an amount of a hyaluronan-associated protein or a functional derivative 
thereof effective for treating wounds; and 
(b) a pharmaceutically acceptable carrier. 
Preferably, the protein has an apparent molecular weight of about 62 kDa on 
SDS PAGE. Most preferably, the protein is calreticulin. 
The above pharmaceutical composition may further comprise 
(c) at least one other agent useful in promoting the healing of a wound. 
Preferred agents include an anti-bacterial agent, an anti-viral agent, an 
anti-fungal agent, a local anesthetic, an analgesic and a growth factor.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present inventors have discovered that hyaluronan in combination with 
its associated proteins (HA-AP), as isolated from tissues, act to promote 
more rapid and relatively scarless wound healing. HA-AP applied topically 
to a wound promotes both the rate of healing as well as the amount of 
scarless healing. As used herein, the term "promote wound healing" is 
intended to include both the rate of healing as well as the diminution of 
scar formation. The term "scarless" as used herein refers both to healing 
with no scar tissue as well as healing with diminished scar formation. 
Also noted by the present inventors was that HA-AP regulates the expression 
of three isoforms of transforming growth factor-.beta. (TGF.beta.). Thus, 
treatment with HA-AP enhanced expression of TGF.beta.3 while inhibiting 
expression of the fibrogenic isoforms TGF.beta.1 and TGF.beta.2, as 
detected with isoform specific antibodies. 
Importantly, the present inventors have discovered that the 62 kDa protein 
associated with HA which is important for scarless wound healing is 
calreticulin, a protein which has been previously described and cloned 
(Fliegel, L. et al. (1989) J. Biol. Chem. 264:21522-21528; Baksh, S. et 
al., (1991) J. Biol. Chem. 266:21458-21465; Rokeach, L. A. et al., (1991) 
Prot. Engineering 4:981-987; Baksh, S. et al. (1992) Prot. Express. 
Purific. 3:322-331; Michalak, M. et al., (1992) Biochem. J. 285:681-692). 
Calreticulin was originally identified as an intra-endoplasmic reticulum 
low affinity, high capacity calcium-binding protein found in non-skeletal 
muscle cells which shows remarkable homology between mammalian species. 
Thus the present invention is directed to methods for promoting the 
scarless healing of a wound comprising administering an effective amount 
of HA-AP, more preferably, a 62 kDa protein of HA-AP, most preferably, 
calreticulin. In these methods, functional derivatives of the above 
protein or proteins may also be administered. Also provided are 
pharmaceutical compositions comprising HA-AP, more preferably a 62 kDa 
protein of HA-AP, most preferably, calreticulin, or a functional 
derivative thereof. 
Proteins, Peptides and Their Functional Derivatives 
The present invention is directed to compositions and methods for promoting 
scarless healing of a wound using HA-AP as well as calreticulin. Also 
included are peptides or other functional derivatives of the HA-associated 
protein or of calreticulin which have the functional activity of promoting 
accelerated and scarless wound healing. 
It will be understood that the protein useful in the methods and 
compositions of the present invention can be biochemically purified from a 
cell or tissue source. For preparation of naturally occurring HA-AP or 
calreticulin, any of a number of tissues of adult or of fetal origin can 
be used. Methods for purifying HA-AP are well-known in the art. See, for 
example, DePalma et al., 1989, supra; Longaker et al. (1989) supra; (Swann 
et al. (1975) supra; Burd et al. (1989) Matrix 9:322-327, which references 
are hereby incorporated by reference in their entirety). 
Alternatively, because the gene encoding calreticulin is known (Fliegel et 
al., supra; Baksh et al., (1991) supra; Rokeach et al., supra; Baksh et 
al. (1992) supra; Michalak et al., supra) and can be isolated or 
synthesized, the polypeptide can be synthesized substantially free of 
other proteins or glycoproteins of mammalian origin in a prokaryotic 
organism or in a non-mammalian eukaryotic organism, if desired. 
Alternatively, methods are well known for the synthesis of polypeptides of 
desired sequence on solid phase supports and their subsequent separation 
from the support. 
Preparation of HA-AP and Calreticulin 
Tissue Extraction 
Frozen fetal sheep skin is thawed at room temperature and multiple 
specimens from time dated ewes are pooled and finely minced into 0.5 cm 
pieces using scissors. The intracellular and extracellular components of 
the skin matrix are extracted using a solution of 4M guanidine-HCl 
containing proteinase inhibitors. Four ml of buffer are used per gram of 
wet fetal skin. The mixture is gently stirred for 72 hours at 4.degree. C. 
Larger pieces of skin are separated from the extract using cheesecloth. To 
maximize yield, the extraction procedure may be repeated. The extract is 
centrifuged for 30 min at 36,000 rpm using a 60Ti Beckman rotor. The 
supernatant is dialyzed against distilled water until free of 
guanidine-HCl when tested with 1% silver nitrate. The extract is then 
resuspended in a solution of 7M urea and 0.15M Tris (Buffer N) titrated to 
a pH of 7.5 using HCl. The resuspended extract is again centrifuged as 
above until all solid and undissolved particles are removed. 
Anion Exchange Chromatography 
Anion exchange chromatography using diethylaminoethyl cellulose (DEAE 52), 
a technique well-known in the art, is used to fractionate the extract. The 
extract is loaded on a DEAE column and washed with 4 time the column 
volume with Buffer N. An increasing gradient of from 0.2M to 1.0M NaCl in 
buffer N is used to separate the extract into fractions of unbound 
protein, collagen, hyaluronic acid and sulfated glycosaminoglycans. These 
fractions are analyzed for uronic acid content using the Carbazole 
reaction. Absorbance at 530 nm are used to detect uronic acid 
concentration. Absorbances at 280 nm are used to measure protein. Based on 
the elution profile, samples are pooled and dialyzed against water until 
free of urea. Samples are lyophilized. Impure or mixed samples containing 
sulfated and nonsulfated glycosaminoglycans are further purified using 
smaller (15-30 ml) anion exchange column as above. 
Alcohol Precipitation 
Alcohol precipitation with ethanol is used to partially purify samples 
which cannot be purified by anion exchange chromatography. The samples are 
dissolved in 4M guanidine HCl solution. Ethyl alcohol is added to achieve 
a 25% alcohol guanidine mixture and the solution centrifuged at 36,000 rpm 
for 45 minutes. The supernatant is preserved an resuspended to a 50% 
ethanol concentration. The pellet is dissolved in 1M NaCl, dialyzed 
against water, and lyophilized. This procedure is then repeated with 
increasing ethanol concentrations of 50% and 75%. 
Cellulose Acetate Electrophoresis 
Purity of the samples is confirmed by cellulose acetate electrophoresis. 
Dry sample are resuspended in water, and 1-2 .mu.l aliquots are placed on 
cellulose acetate plates. Standard samples containing sulfated and 
non-sulfated glycosaminoglycans are place on either side of the test 
samples. Cellulose acetate plates are stained with 1% alcian blue stain 
and destained with 5% acetic acid. This method detects polysaccharides of 
MW &gt;7.2 kDa and is sensitive to 0.1 .mu.g of glycosaminoglycans. 
Purified samples of hyaluronic acid are suspended in buffer containing 
0.15M NaCl and 0.1M Sodium acetate titrated to pH 5. The samples are 
treated with hyaluronidase and incubated at 60.degree. C. for 4 hours. 
Disappearance of the characteristic hyaluronic acid bands on cellulose 
acetate electrophoresis confirms the purity of the samples. 
Polyacrylamide Gel Electrophoresis (PAGE) 
PAGE is performed sing a 5%-20% gradient resolving gels and a 3.5% stacking 
gel to demonstrate the presence of the HA-APs. The preferred technique is 
a modification of the method of D. A. Swann et al. (1983) J. Biol. Chem. 
258:2683-2688. Highly purified dried samples of fetal HA and its 
associated protein are dissolved in reducing buffer to disrupt interchain 
disulfide bonds. The samples are boiled to break protein aggregates. 
Successively increasing concentrations of sample are applied to the gels 
along with high and low MW standards used as references until protein 
banding is demonstrated. Gels are stained with 1% coomassie blue and 
destained with methanol-acetic acid in distilled water. 
Hyaluronic Acid Content of Samples 
The uronic acid content of samples is quantitated using the carbazole 
reaction. Standard samples of HA derived from rooster comb are analyzed. 
Absorbance is measured at 530 nm. 
Protein concentration is measured using the Lowry method, with BSA as 
standard. 
Microanalysis of SDS-PAGE Electroblotted Protein 
The current availability of chemically stable membranes provides the means 
for direct sequencing of peptides after being electroblotted in microgram 
quantities, for example from a SDS gel. Aebersold et al., developed such a 
PAGE electroblotting method to isolate microgram quantities of protein for 
amino acid sequence analysis. This system also offers a means of purifying 
the protein to be sequenced by differential mobilities based on the 
protein MW. In the present methods, extracted fetal HA-AP, for example 
from 90 days of gestation is electroblotted to nylon PVDF (Immobilon; 
Millipore) membranes. The proteins are transferred for 1.5 hours and 
stained for 30 seconds with 0.1% fast Green, 10% glacial acetic acid, and 
25% methanol, and destained in 5% acetic acid for 1 minute, and washed 
profusely with distilled water for 20 minutes. The membranes are then 
encased in plastic wrap and the sequence determinations performed. This 
process is repeated for each band on SDS-PAGE. Available protein and DNA 
databases are used to identify the sequenced protein. If the protein is 
blocked at its N-terminus, internal sequence analysis is performed by a 
technique based on CNBr cleavage and ortho-phthaldehyde blocking of the 
N-terminus of fragments not containing proline. The biological activity 
and dose response of proteins purified, identified and sequenced in this 
way will be tested as described below. 
The above method was used to identify the 62 kDa HA-AP as having identical 
amino acid sequence with calreticulin. 
In a further embodiment, the invention provides "functional derivatives" of 
a HA-AP, particularly, of calreticulin. By "functional derivative" is 
meant a "fragment," "variant," "analog," or "chemical derivative" of 
calreticulin. A functional derivative retains at least a portion of the 
function of calreticulin, such as the activity of promoting scarless wound 
healing, upregulating TGF.beta.3 expression in skin, or binding to a 
specific anti-calreticulin antibody, which permits its utility in 
accordance with the present invention. 
A "fragment" of calreticulin refers to any subset of the molecule, that is, 
a shorter peptide. 
A "variant" of calreticulin refers to a molecule substantially similar to 
either the entire protein or a fragment thereof. Variant peptides may be 
conveniently prepared by direct chemical synthesis of the variant peptide, 
using methods well-known in the art. 
Alternatively, amino acid sequence variants of the protein or peptide can 
be prepared by mutations in the DNA which encodes the synthesized peptide. 
Such variants include, for example, deletions from, or insertions or 
substitutions of, residues within the amino acid sequence. Any combination 
of deletion, insertion, and substitution may also be made to arrive at the 
final construct, provided that the final construct possesses the desired 
functional activity. Obviously, the mutations that will be made in the DNA 
encoding the variant peptide must not alter the reading frame and 
preferably will not create complementary regions that could produce 
secondary mRNA structure (see European Patent Publication No. EP 75,444). 
At the genetic level, these variants ordinarily are prepared by 
site-directed mutagenesis (as exemplified by Adelman et al., DNA 2:183 
(1983)) of nucleotides in the DNA encoding the calreticulin protein or a 
peptide fragment thereof, thereby producing DNA encoding the variant, and 
thereafter expressing the DNA in recombinant cell culture (see below). The 
variants typically exhibit the same qualitative biological activity as the 
nonvariant peptide. 
A preferred group of variants of calreticulin are those in which at least 
one amino acid residue in the protein or in a peptide fragment thereof, 
and preferably, only one, has been removed and a different residue 
inserted in its place. For a detailed description of protein chemistry and 
structure, see Schulz, G. E. et al., PRINCIPLES OF PROTEIN STRUCTURE, 
Springer-Verlag, New York, 1978, and Creighton, T. E., PROTEINS: STRUCTURE 
AND MOLECULAR PROPERTIES, W. H. Freeman & Co., San Francisco, 1983, which 
are hereby incorporated by reference. The types of substitutions which may 
be made in the protein or peptide molecule of the present invention may be 
based on analysis of the frequencies of amino acid changes between a 
homologous protein of different species, such as those presented in Table 
1-2 of Schulz et al. (supra) and FIGS. 3-9 of Creighton (supra). Base on 
such an analysis, conservative substitutions are defined herein as 
exchanges within one of the following five groups: 
1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr 
(Pro, Gly); 
2. Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gln; 
3. Polar, positively charged residues: His, Arg, Lys; 
4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys); and 
5. Large aromatic residues: Phe, Tyr, Trp. 
The three amino acid residues in parentheses above have special roles in 
protein architecture. Gly is the only residue lacking any side chain and 
thus imparts flexibility to the chain. Pro, because of its unusual 
geometry, tightly constrains the chain. Cys can participate in disulfide 
bond formation which is important in protein folding. Note the Schulz et 
al. would merge Groups 1 and 2, above. Note also that Tyr, because of its 
hydrogen bonding potential, has some kinship with Ser, Thr, etc. 
Substantial changes in functional or immunological properties are made by 
selecting substitutions that are less conservative, such as between, 
rather than within, the above five groups, which will differ more 
significantly in their effect on maintaining (a) the structure of the 
peptide backbone in the area of the substitution, for example, as a sheet 
or helical conformation, (b) the charge or hydrophobicity of the molecule 
at the target site, or (c) the bulk of the side chain. Examples of such 
substitutions are (a) substitution of gly and/or pro by another amino acid 
or deletion or insertion of gly or pro; (b) substitution of a hydrophilic 
residue, such as ser or thr, for (or by) a hydrophobic residue, such as 
leu, ile, phe, val or ala; (c) substitution of a cys residue for (or by) 
any other residue; (d) substitution of a residue having an electropositive 
side chain, such as lys, arg or his, for (or by) a residue having an 
electronegative charge, such as glu or asp; or (e) substitution of a 
residue having a bulky side chain, such as phe, for (or by) a residue not 
having such a side chain, such as gly. 
Preferred deletions and insertions, and substitutions, according to the 
present invention, are those which do not produce radical changes in the 
characteristics of the protein or peptide molecule. However, when it is 
difficult to predict the exact effect of the substitution, deletion, or 
insertion in advance of doing so, one skilled in the art will appreciate 
that the effect will be evaluated by routine screening assays which are 
described in more detail below. For example, a change in the immunological 
character of the protein peptide molecule, such as binding to a given 
antibody, is measured by a competitive type immunoassay. Biological 
activity is screened in an appropriate bioassay, as described below. 
Modifications of such peptide properties as redox or thermal stability, 
hydrophobicity, susceptibility to proteolytic degradation or the tendency 
to aggregate with carriers or into multimers are assayed by methods well 
known to the ordinarily skilled artisan. 
An "analog" of calreticulin refers to a non-natural molecule substantially 
similar to either the entire molecule or a fragment thereof. 
A "chemical derivative" of calreticulin contains additional chemical 
moieties not normally a part of the peptide. Covalent modifications of the 
peptide are included within the scope of this invention. Such 
modifications may be introduced into the molecule by reacting targeted 
amino acid residues of the peptide with an organic derivatizing agent that 
is capable of reacting with selected side chains or terminal residues. 
Additionally, modified amino acids or chemical derivatives of amino acids 
of calreticulin or fragments thereof, according to the present invention 
may be provided, which polypeptides contain additional chemical moieties 
or modified amino acids not normally a part of the protein. Covalent 
modifications of the peptide are thus included within the scope of the 
present invention. The following examples of chemical derivatives are 
provided by way of illustration and not by way of limitation. 
Aromatic amino acids may be replaced with D- or L-naphthylalanine, D- or 
L-phenylglycine, D- or L-2-thienylalanine, D- or L-1-, 2-, 3- or 
4-pyrenylalanine, D- or L-3-thienylalanine, D- or L-(2-pyridinyl)-alanine, 
D- or L-(3-pyridinyl)-alanine, D- or L-(2-pyrazinyl)-alanine, D- or 
L-(4-isopropyl)-phenylglycine, D-(trifluoromethyl)-phenylglycine, 
D-(trifluoromethyl)-phenylalanine, D-p-fluorophenylalanine, D- or 
L-p-biphenylphenylalanine, D- or L-p-methoxybiphenylphenylalanine, D- or 
L-2-indole(alkyl)alanine, and D- or L-alkylalanine where alkyl may be 
substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, 
isopropyl, isobutyl, sec-isotyl, isopentyl, non-acidic amino acids, of 
chain lengths of C1-C20. 
Acidic amino acids can be substituted with non-carboxylate amino acids 
while maintaining a negative charge, and derivatives or analogs thereof, 
such as the non-limiting examples of (phosphono)-alanine, glycine, 
leucine, isoleucine, threonine, or serine; or sulfated (for example, 
--SO.sub.3 H) threonine, serine, tyrosine. 
Other substitutions may include unnatural hydroxylated amino acids may made 
by combining "alkyl" with any natural amino acid. Basic amino acids may be 
substituted with alkyl groups at any position of the naturally occurring 
amino acids lysine, arginine, ornithine, citrulline, or (guanidino)-acetic 
acid, or other (guanidino)alkyl-acetic acids, where "alkyl" is define as 
above. Nitrile derivatives (for example, containing the CN-moiety in place 
of COOH) may also be substituted for asparagine or glutamine, and 
methionine sulfoxide may be substituted for methionine. Methods of 
preparation of such peptide derivatives are well known to one skilled in 
the art. 
In addition, any amide linkage the polypeptides can be replaced by a 
ketomethylene moiety, for example, (--C(.dbd.O)--CH.sub.2 --) for 
(--(C.dbd.O)--NH--). Such derivatives are expected to have the property of 
increased stability to degradation by enzymes, and therefore possess 
advantages for the formulation of compounds which may have increased in 
vivo half lives, as administered by various routes as described herein. 
In addition, any amino acid representing a component of the peptides can be 
replaced by the same amino acid but of the opposite chirality. Thus, any 
amino acid naturally occurring in the L-configuration (which may also be 
referred to as the R or S, depending upon the structure of the chemical 
entity) may be replaced with an amino acid of the same chemical structural 
type, but of the opposite chirality, generally referred to as the D-amino 
acid but which can additionally be referred to as the R- or the S-, 
depending upon its composition and chemical configuration. Such 
derivatives have the property of greatly increased stability to 
degradation by enzymes, and therefore are advantageous in the formulation 
of compounds which may have longer in vivo half lives, when administered 
by various routes. 
Additional amino acid modifications in calreticulin or in a peptide thereof 
may include the following. 
Cysteinyl residues most commonly are reacted with .alpha.-haloacetates (and 
corresponding amines), such as chloroacetic acid or chloroacetamide, to 
give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues 
also are derivatized by reaction with bromotrifluoroacetone, 
.alpha.-bromo-.beta.-(5-imidozoyl)propionic acid, chloroacetyl phosphate, 
N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl 
disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or 
chloro-7-nitrobenzo-2-oxa-1,3-diazole. 
Histidyl residues are derivatized by reaction with diethylprocarbonate at 
pH 5.5-7.0 because this agent is relatively specific for the histidyl side 
chain. Para-bromophenacyl bromide also is useful; the reaction is 
preferably performed in 0.1M sodium cacodylate at pH 6.0. 
Lysinyl and amino terminal residues are reacted with succinic or other 
carboxylic acid anhydrides, which reverses the charge of the lysinyl 
residues. Other suitable reagents for derivatizing 
.alpha.-amino-containing residues include imidoesters such as methyl 
picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; 
trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and 
transaminase-catalyzed reaction with glyoxylate. 
Arginyl residues are modified by reaction with one or several conventional 
reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, 
and ninhydrin. Derivatization of arginine residues requires that the 
reaction be performed in alkaline conditions because of the high pK.sub.a 
of the guanidine functional group. Furthermore, these reagents may react 
with the groups of lysine as well as the arginine .epsilon.-amino group. 
The specific modification of tyrosyl residues has been studied extensively 
with particular interest in introducing spectral labels into tyrosyl 
residues by reaction with aromatic diazonium compounds or 
tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane 
are used to form O-acetyl tyrosyl species and 3-nitro derivatives, 
respectively. 
Carboxyl side groups (aspartyl or glutamyl) are selectively modified by 
reaction with carbodiimides (R'-N-C-N-R') such as 
1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 
1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, 
aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl 
residues by reaction with ammonium ions. 
Glutaminyl and asparaginyl residues are deamidated to the corresponding 
glutamyl and aspartyl residues. Alternatively, these residues are 
deamidated under mildly acidic conditions. Either form of these residues 
falls within the scope of this invention. 
Derivatization with bifunctional agents is useful for cross-linking the 
peptide to a water-insoluble support matrix or to other macromolecular 
carriers. Commonly used cross-linking agents include, e.g., 
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide 
esters, for example, esters with 4-azidosalicylic acid, homobifunctional 
imidoesters, including disuccinimidyl esters such as 
3,3'-dithiobis-(succinimidyl-propionate), and bifunctional maleimides such 
as bis-N-maleimido-1,8-octane. Derivatizing agents such as 
methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable 
intermediates that are capable of forming crosslinks in the presence of 
light. Alternatively, reactive water-insoluble matrices such as cyanogen 
bromide-activated carbohydrates and the reactive substrates described in 
U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 
4,330,440 are employed for protein immobilization. 
Other modifications include hydroxylation of proline and lysine, 
phosphorylation of hydroxyl groups of seryl or threonyl residues, 
methylation of the .alpha.-amino groups of lysine, arginine, and histidine 
side chains (Creighton, supra), acetylation of the N-terminal amine, and, 
in some instances, amidation of the C-terminal carboxyl groups. 
Such derivatized moieties may improve the solubility, absorption, 
biological half life, and the like. The moieties may alternatively 
eliminate or attenuate any undesirable side effect of the protein and the 
like. Moieties capable of mediating such effects are disclosed, for 
example, in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing 
Co., Easton, Pa. (1980). 
Production of Calreticulin and Fusion Proteins that Promote Scarless Wound 
Healing 
Calreticulin may be purified from a tissue source using conventional 
biochemical techniques, or produced recombinantly in either prokaryotic or 
eukaryotic cells using methods well-known in the art (Sambrook, J. et al., 
MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Edition, Cold Spring Harbor 
Press, Cold Spring Harbor, N.Y., 1989, which reference is hereby 
incorporated by reference in its entirety). Various references describing 
the cloning and expression of calreticulin have been noted above. 
Fusion proteins representing different polypeptide regions in calreticulin 
may be used to identify regions of the protein that have the desired 
functional activity (binding, stimulating wound healing, etc.). When 
combined with the polymerase chain reaction (PCR) method, it is possible 
and expedient to express in bacteria nearly any selected region of the 
protein. 
To facilitate unidirectional subcloning of the PCR products, sense and 
antisense oligonucleotides have been designed to include BamH1 recognition 
sequences at the 5' end and EcoR1 recognition sequences at the 3' end, 
respectively; appropriately digested PCR products are then be ligated 
directly into a vector (such as the pGEX-2T vector). Use of this 
methodology allows construction of vectors and purification of several 
fusion proteins in less than one month. 
The pGEX vector is preferred because the glutathione-S-transferase (GST) 
fusion proteins can be purified rapidly by binding to glutathione-agarose 
beads. In addition, because cDNAs are cloned into pGEX-2T, the portion of 
the fusion protein representing the GST can be cleaved with thrombin and 
the engineered polypeptide can generally be recovered free of the GST 
protein which can be removed using glutathione-agarose beads (Ausubel, F. 
M., et al., 1990, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & 
Sons, New York. 
Calreticulin, a peptide thereof, or a fusion protein thereof may also be 
expressed in insect cells using baculovirus expression system. Production 
of calreticulin or functional derivatives thereof, including fusion 
proteins, in insects can be achieved, for example, by infecting the insect 
host with a baculovirus engineered to express calreticulin by methods 
known to those of skill. Thus, in one embodiment, sequences encoding 
calreticulin may be operably linked to the regulatory regions of the viral 
polyhedrin protein (Jasny, 1987, Science 238:1653). Infected with the 
recombinant baculovirus, cultured insect cells, or the live insects 
themselves, can produce the calreticulin or functional derivative protein 
in amounts as great as 20 to 50% of total protein production. When live 
insects are to be used, caterpillars are presently preferred hosts for 
large scale production according to the invention. 
Fragments of calreticulin are purified by conventional affinity 
chromatography using antibodies, preferably monoclonal antibodies (mAbs), 
that recognize the appropriate regions of calreticulin. The mAbs specific 
for the most highly conserved regions in calreticulin can be used to 
purify calreticulin protein from mixtures. 
Wound Healing Assays 
To characterize functions of HA-AP, of calreticulin, and of different 
regions in calreticulin, any of a number of assays may be used. These 
assays may be used routinely to analyze the biological functions of 
calreticulin or other HA-AP of the present invention. 
Fibroblast Assays 
A. Fibroblast Proliferation 
Normal human dermal fibroblast cultures are maintained in a humidified 5% 
CO.sub.2 incubator at 37.degree. C. Preferred medium is Dulbecco's 
modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum 
and antibiotics. The presence of fetal bovine serum at this concentration 
has no effect on HA-AP activity. Cultures are established in 35 mm dishes 
with 10.sup.5 cells/dish in 2 ml medium. The test preparation, for 
example, HA-AP or calreticulin, is added at the time of plating or 24 
hours later. Cultures are pulsed with .sup.3 H-thymidine for 24 hours 
beginning at 24 or 48 hours of culture. Cells are harvested at 48 or 72 
hours using trypsin, counted and isotope incorporation is measured by 
liquid scintillation counting. DNA content in the culture is also 
measured. Since fibroblasts are the cells responsible for matrix 
accumulation in scarring, it is expected that the agents active in 
promoting scarless wound healing would inhibit fibroblast proliferation. 
B. Fibroblast Motility 
Fibroblast-populated collagen lattice (FPCL (Bell, E. et al., (1979) Proc. 
Natl. Acad. Sci. U.S.A. 76:1274) is composed of soluble collagen, cultured 
fibroblasts and serum-enriched culture medium, which are rapidly mixed. 
The collagen polymerizes, entrapping cells within it. Over time, the cells 
within the lattice cause a reorganization of the matrix, with condensation 
and local alignment of collagen fibrils. The overall effect is the 
shrinkage of the lattice, referred to as "lattice contraction. Acid 
soluble rat tail tendon and pepsin solubilized human leiomyoma collagens 
are isolated by salt precipitation. After extensive dialysis, the collagen 
solutions are frozen, lyophilized and resuspended in sterile 1 mM HCl at 5 
mg/ml and stored at 4.degree. C. Standard lattice preparation involves 
gentle vortex mixing of 1 ml of culture medium, 0,5 ml of cells and 0.5 m 
of collagen solution, and rapid pouring of the mixture into 35 mm dishes 
which are incubated at 37.degree. C. HA-AP or calreticulin preparations 
are added either (a) at the time of lattice formation, (b) after 
polymerization, or (c) at 24 hrs. The lattices are measured using 
computerized morphometrics to give an area of the lattice for each 12 
hours, and the rate of contraction is recorded. The rate of contraction is 
determined for the varying doses of test agents applied. It is expected 
that the wound healing promoting compounds of the present invention 
increase contraction. 
C. Fibroblast Metabolism 
Comprehensive analysis of collagen metabolism is performed with and without 
the various test compounds, using double labeling techniques (Bateman, J. 
F. et al., (1988) Anal. Biochem. 168:171-175). Confluent fibroblast 
monolayer cultures are incubated with a mixture of D-[4-.sup.14 C]-proline 
and L-[4-.sup.3 H-proline in medium with and without fetal bovine serum. 
Following incubation, the cell layer and medium fractions are treated 
separately. Collagens in the medium are precipitated with ammonium sulfate 
and resuspended in 50 mM Tris-HCl pH 7.5, containing 0.15M NaCl and 
proteinase inhibitors. Procollagen is precipitated by addition of ethanol 
and converted to .alpha.-chains by limited pepsin digestion. The cell 
fraction is sonicated and centrifuged. DNA analysis is performed on half 
of the solution and collagen precipitated from the other half. Again, 
procollagen is isolated and .alpha.-chains produced by limited pepsin 
digestion. 
Collagen production and secretion, and proline hydroxylation is analyzed by 
the incorporation of 14C-protein into bacterial collagenase-digestible 
protein and assessing changes in the 3H:14C proline ratios. The production 
and secretion of individual types of collagen is assessed by incorporation 
of 14C proline into individual a-chains, separated by gel electrophoresis, 
and quantified by liquid scintillation counting of excised portions of the 
gel. Collagen production/secretion experiments are also done by using one 
radiolabeled precursor in culture and determining the hydroxyproline 
contents. 
Re-epithelization Assays in a Dermal Explant Model 
A. Epidermal Cell Proliferation 
Dermal sheets from paravertebral areas of domestic pig skin are obtained 
under aseptic conditions with a Pagett's dermatome at a setting of 0.5 mm 
after removing a 0.5 mm thick split thickness graft containing the 
epidermis. Dermal strips are cut into 1 cm2 segments and transferred onto 
stile dressing sponges in multiwell Petri dishes to raise the explant to 
the air liquid interface. Orientation of the dermis is maintained. 
Explants are kept in a 10%CO2 environment with 95% humidity in serum free 
DME supplemented by 10 ng/ml hydrocortisone without antibiotics. 
The test compound is suspended in sterile PBS at various concentrations and 
applied to the explant (75 .mu./cm2) at day o only or at the time of 
medium exchanges every 4 days. Explant cultures are harvested after 4, 8 
or 12 days and fixed in 10% formalin. Re-epithelialized areas around hair 
follicles are visualized by staining with 1% rhodamine solution for a few 
seconds and washing in formalin. Resurfaced areas are photographed at a 
magnification of 10.times. and their size determined by computerized 
morphometric analysis. The ration of area of re-epithelialization to the 
cross sectional area of the hair follicle is determined. 
Full Thickness in vivo Wound Healing Models 
Full thickness paired 1 cm incisional and paired 1 cm2 excisional wounds 
are made with a sterile scalpel on the dorsa of male Sprague-Dawley rats. 
The test compound, suspended in PBS, is injected into the incisional wound 
margins or dissolved in a vehicle such as 70:30 lanolin petrolatum with 
proteinase inhibitor and applied topically to the excisional wounds. The 
test compound is applied daily to an incisional and excisional wound on 
each animal. The paired wound is treated daily with the corresponding 
control (PBS alone or lanolin-petrolatum vehicle). Animals are housed 
individually and their wounds are covered daily. Treatment is randomized 
to right or left side independently for incisional and excisional wounds. 
Wound closure is determined by tracing the excisional wounds on acetate 
sheets and measuring their areas using computer assisted planimetry. 
Animals are sacrificed at specified intervals after wounding and the 
tissue fixed overnight i 10% buffered formalin and embedded in paraffin. 
Hematoxylin and eosin staining of section is used to count inflammatory 
cells at wound margins per high power filed. Masson's trichrome stained 
slides are graded for collagen organization based on the thickness and 
orientation of collagen fibers and the extent of scar present. 
Immunohistochemistry is performed using various antibodies as described 
herein, including TGF.beta. isoform-specific antibodies, anti-fibronectin 
antibodies and antibodies to collagen types I and III. After relative 
effects of a given protein, peptide, etc. on wound healing is determined, 
dose response relationships are obtained using the above model. 
In the above assays, wounds treated with a sample containing a HA-AP having 
activity for promoting healing, or having calreticulin, heal more quickly. 
Specificity may be tested by including an anti-calreticulin antibody or an 
antibody against another HA-AP in the treatment mixture. If an 
anti-calreticulin antibody inhibits the bioactivity of the test sample, 
the activity can be attributed to calreticulin. 
Therapeutic Applications of Hyaluronan-Associated Proteins and Calreticulin 
The preferred animal subject of the present invention is a mammal. By the 
term "mammal" is meant an individual belonging to the class Mammalia. The 
invention is particularly useful in the treatment of human subjects. 
The present invention provides for methods of treatment of wounds, which 
methods comprise administering to a subject in need of such treatment an 
effective amount of an HA-AP, preferably a 62 kDa protein of HA-AP, most 
preferably, calreticulin, or a functional derivative thereof, that promote 
the scarless healing of a wound. 
The disorders that may be treated according to this invention include, but 
are not limited to surgical wounds, wounds incurred in accidents, or 
wounds associated with any of a number of diseases including cancer and 
infectious disease. 
Effective doses of calreticulin for therapeutic uses discussed above may be 
determined using methods known to one skilled in the art. Effective doses 
may be determined, preferably in vitro, in order to identify the optimal 
dose range using various of the methods described herein. In one 
embodiment, an aqueous solution of a calreticulin protein or peptide is 
administered by intravenous injection. Each dose may range from about 
0.001 .mu.g/kg body weight to about 100 mg/kg body weight, or more 
preferably, from about 0.1 .mu.g/kg to 10 mg/kg body weight. The dosing 
schedule may vary from once a week to daily depending on a number of 
clinical factors, including the type of wound, its severity, and the 
subject's sensitivity to the protein. Nonlimiting examples of dosing 
schedules are 3 .mu.g/kg administered twice a week, three times a week or 
daily; a dose of 7 .mu.g/kg twice a week, three times a week or daily; a 
dose of 10 .mu.g/kg twice a week, three times a week or daily; or a dose 
of 30 .mu.g/kg twice a week, three times a week or daily. In the case of a 
more severe wound, it may be preferable to administer doses such as those 
described above by alternate routes, including intravenously or 
intrathecally. Continuous infusion may also be appropriate. 
Calreticulin or a functional derivative may also be administered in 
combination with an effective amount of at least one other agent that is, 
itself, capable of promoting the healing of wounds or treating 
accompanying symptoms. Such agents include growth factors, 
anti-infectives, including anti-bacterial, anti-viral and anti-fungal 
agents, local anesthetics, and analgesics, or a combination thereof. 
The calreticulin may be administered in any pharmaceutically acceptable 
carrier. The administration route may be any mode of administration known 
in the art, including but not limited to intravenously, intrathecally, 
subcutaneously, or intracranially by injection into involved tissue, 
intraarterially, orally, or via an implanted device. 
The present invention also provides pharmaceutical compositions comprising 
an amount of a HA-AP, preferably a 62 kDa protein of an HA-AP, most 
preferably, calreticulin, or a functional derivative thereof effective to 
promote the scarless healing of a wound, in a pharmaceutically acceptable 
carrier. 
Also provided is a pharmaceutical composition comprising an effective 
amount of calreticulin together with one or more additional agents in a 
pharmaceutically acceptable carrier. Such additional agents include agents 
which are known to promote wound healing or to treat problems or symptoms 
associated with wounds. Examples of such agents include disinfectants such 
as antibacterial agents or antiviral agents, anti-fungal agents, 
anti-inflammatory agents, agents which induce relief from pain or itching, 
and the like. Also including are growth factors which promote wound 
healing, including, but not limited to, transforming growth 
factor-.alpha., transforming growth factor-.beta., fibroblast growth 
factor-.alpha., fibroblast growth factor-.beta., epidermal growth factor, 
platelet-derived growth factor, endothelial cell-derived growth factor, 
insulin-like growth factors, and granulocyte colony-stimulating factor. 
The pharmaceutical compositions of the present invention may be 
administered by any means that achieve their intended purpose. Amounts and 
regimens for the administration of a HA-AP, calreticulin, or a derivative 
thereof, can be determined readily by those with ordinary skill in the 
clinical art of treating wounds. 
The pharmaceutical composition of the present invention is preferably 
applied topically to a wound. For topical application, the compositions of 
the present invention may be incorporated into topically applied vehicles 
such as salves or ointments, which have both a soothing effect on the skin 
as well as a means for administering the active ingredient directly to the 
affected area. 
The carrier for the active ingredient may be either in sprayable or 
nonsprayable form. Non-sprayable forms can be semi-solid or solid forms 
comprising a carrier indigenous to topical application and having a 
dynamic viscosity preferably greater than that of water. Suitable 
formulations include, but are not limited to, solution, suspensions, 
emulsions, creams, ointments, powders, liniments, salves, and the like. If 
desired, these may be sterilized or mixed with auxiliary agents, e.g., 
preservatives, stabilizers, wetting agents, buffers, or salts for 
influencing osmotic pressure and the like. Preferred vehicles for 
non-sprayable topical preparations include ointment bases, e.g., 
polyethylene glycol-1000 (PEG-1000); conventional creams such as HEB 
cream; gels; as well as petroleum jelly and the like. A most preferred 
vehicle is a petrolatum/lanolin vehicle. 
Also suitable for topic application are sprayable aerosol preparations 
wherein the active ingredient, preferably in combination with a solid or 
liquid inert carrier material, is packaged in a squeeze bottle or in 
admixture with a pressurized volatile, normally gaseous propellant. The 
aerosol preparations can contain solvents, buffers, surfactants, perfumes, 
and.or antioxidants in addition to the compounds of the invention. 
For the preferred topical applications, it is preferred to administer an 
effective amount of a composition according to the present invention to an 
affected wound area, in particular the skin surface. This amount will 
generally range from about 0.001 mg to about 1 g per application, 
depending upon the area to be treated, the severity of the symptoms, and 
the nature of the topical vehicle employed. A preferred topical 
preparation is an ointment wherein about 0.01 to about 50 mg of active 
ingredient is used per cc of ointment base. 
Alternatively, or concurrently, administration may be by parenteral, 
subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or 
buccal routes. Alternatively, or concurrently, administration may be by 
the oral route. The dosage administered will be dependent upon the age, 
health, and weight of the recipient, kind of concurrent treatment, if any, 
frequency of treatment, and the nature of the effect desired. 
Compositions within the scope of this invention include all compositions 
wherein the protein, peptide or derivative is contained in an amount 
effective to achieve its intended purpose. While individual needs vary, 
determination of optimal ranges of effective amounts of each component is 
within the skill of the art. Typical dosages comprise 0.01 to 100 
mg/kg/body wt. The preferred dosages comprise 1 to 100 mg/kg/body wt. 
In addition to the pharmacologically active compounds, the new 
pharmaceutical preparations may contain suitable pharmaceutically 
acceptable carriers comprising excipients and auxiliaries which facilitate 
processing of the active compounds into preparations which can be used 
pharmaceutically. Suitable formulations for oral administration include 
hard or soft gelatin capsules, dragees, pills tablets, including coated 
tablets, elixirs, suspensions, syrups or inhalations and controlled 
release forms thereof. Preparations which can be administered rectally are 
suppositories. Suitable injectable solutions include intravenous 
subcutaneous and intramuscular injectable solutions. The compositions may 
also be administered in the form of an infusion solution or as a nasal 
inhalation or spray. Suitable solutions for administration by B injection 
or orally, contain from about 0.01 to 99 percent, preferably from about 20 
to 75 percent of active compound(s), together with the excipient. The 
pharmaceutical formulation for systemic administration according to the 
invention may be formulated for enteral, parenteral or topical 
administration. Indeed, all three types of formulation may be used 
simultaneously to achieve systemic administration of the active 
ingredient. 
Having now generally described the invention, the same will be more readily 
understood through reference to the following examples which are provided 
by way of illustration, and are not intended to be limiting of the present 
invention, unless specified. 
EXAMPLE I 
Hyaluronan-Associated Protein in Fetal Sheep Skin 
Using the fetal sheep model, the present inventors were able to obtain a 
large amount of fetal skin over a long gestational period (Term=145 days). 
As discussed previously, a large difference was found in the concentration 
of fetal skin hyaluronan between the "scarless period" when HA 
concentrations are high and the typical post-natal scarred healing period. 
Similarly, the present inventors' laboratories characterized the 
hyaluronan associated proteins of different gestational ages (Bakshandeh, 
N. et al. (1992) Biochem Intl 28:843-851). 
The total protein associated with HA decreased from 42% of the dry weight 
at 75 days gestation when scarless healing predominates to its low of 22% 
at 125 days gestation scarred healing begins. Also, the protein associated 
with HA of fetal sheep skin varies in molecular weight depending on its 
gestational age. Specifically, the dominant protein profile changes at 125 
days gestation, when the fetus begins to heal as an adult, from a 62 kDa 
peptide to a smaller protein of about 21 kDa. 
Interestingly, the 62 kDa protein is only found during the period 
coinciding with scarless healing. Whether the 21 kDa peptide represents a 
post-translational modification or a degradation product of the 62 kDa 
protein versus a separate gene product is presently unknown. 
EXAMPLE II 
Dominant 62 kDa Protein Associated with Fetal Sheep Hyaluronan 
Given the above data, the present inventors extracted and highly purified 
early gestational fetal sheep hyaluronan and associated proteins and 
confirmed the 62 kDa fetal hyaluronan associated protein as the dominant 
band in a silver stained SDS-PAGE gel. 
EXAMPLE III 
In Vivo Wound Healing Assay 
The present inventors developed an in vivo wound healing assay in the rat. 
Incisional or excisional wounds were made on the dorsa of rats and were 
treated with HA-AP or corresponding controls. The HA-AP treated wounds 
healed more quickly, with better collagen organization based on light 
microscopy, and an increased cellular response at the wound margin. 
EXAMPLE IV 
Detection of TGF.beta. Isoforms in Wounds 
Antibody Production 
Antibodies against the three mammalian isoforms of TGF.beta. were produced 
by immunization of rabbits with synthetic peptides corresponding to a part 
of each isoform essentially as described (Pelton, R. W. et al., 1991, J. 
Cell Biol. 115:1091-1105). The sequences of each of the three TGF.beta. 
isoforms are identical in mammals. The following amino acid residues were 
used: TGF.beta.1 and TGF.beta.2, residues 4-19; TGF.beta.3, residues 9-20. 
The peptides were synthesized using a 430A peptide synthesizer 
(incorporating the t-boc solid phase synthesis method followed by 
hydrofluoride cleavage. The peptides were purified by HPLC using a 
gradient composed of 0.1% trifluoroacetic acid and 100% acetonitrile. Each 
peptide (5 mg) was dissolved in 0.1M NaHCO.sub.3 and coupled to keyhole 
limpet hemocyanin at a 1:1 (w:w) ratio. Rabbits were initially immunized 
with 500 .mu.g of each peptide and subsequently boosted with 250 .mu.g 
every 2.5 weeks. Antibody titer was determined by ELISA using the 
appropriate uncoupled peptide and alkaline phosphatase-conjugated goat 
anti-rabbit IgG (Promega Biotec, Madison Wis). The three antisera did not 
cross-react with the other two "non-specific" TGF.beta. peptides. Each 
antiserum was purified by ammonium sulfate precipitation (31.3%) followed 
by affinity chromatography using the respective immunogenic peptide. The 
peptide (8 mg) was coupled to 2 ml of Tresyl-Sepharose (Pharmacia) 
overnight according to manufacturer's instructions. The purified IgG was 
eluted with 50 mM glycine (Ph 2.5) into Tris buffer (pH 7.2) for 
neutralization, dialyzed against TBS (0.01M Tris, 0.15M NaCl, pH 8.0), 
aliquoted and stored frozen. 
Western Blot Analysis 
Each anti-peptide antiserum was tested for both immunoreactivity with the 
corresponding mature isoform of the TGF.beta. molecule and for 
cross-reactivity with each other TGF.beta. isoform by Western blot 
analysis. Recombinant human TGF.beta.1 and TGF.beta.3, and native porcine 
TGF.beta.2, were reduced with 0.1M dithiothreitol, subjected to SDS-PAGE 
using a gradient polyacrylamide gel of 10-20% and subsequently transferred 
to nitrocellulose membrane for 1 hr at 1 V using the Biorad Miniblot 
System (Bio-Rad, Cambridge, Mass.). The membranes were blocked with 3% 
nonfat dry milk in TBS for 1 hr and directly incubated overnight in 
purified an anti-peptide IgG preparation in TBS containing 0.1% Tween 20 
(TBST) at dilutions of 1:50 or 1:25. the membranes were washed with TBST 
and incubated with alkaline phosphatase-labeled goat anti-rabbit IgG at a 
dilution of 1:3000 for 1 hr. The blot was developed with the chromogenic 
substrate NBT/BCIP Promega). 
Immunohistochemistry 
Tissues were fixed overnight in 4% paraformaldehyde/phosphate buffered 
saline, dehydrated in increasing concentrations of ethanol and embedded in 
paraffin wax. Sections of 5-7 .mu.m were cut and floated onto coated 
slides. Sections were submerged in TBS/0.1% (v/v) Triton X-100 at room 
temperature for 15 minutes, followed by TBS for 5 min., methanol for 2 
min. and methanol/0.6% (v/v) hydrogen peroxide for 30 min. Slides were 
subsequently washed at room temperature in methanol for 2 min., TBS for 5 
min. and thrice in TBS/0.1% (w/v) bovine serum albumin (BSA) for 3 min. 
After treatment with hyaluronidase (1 mg/ml in 100 mM sodium acetate, 
0.85% NaCl), and three washes in TBS/0.1% BSA, excess protein was blocked 
with 5% normal swine serum in TBS/0.5% BSA for 15 min. at room 
temperature. 
Tissue sections were incubated with primary antibody at a concentration of 
2.5 .mu.g/ml overnight at 4.degree. C. C.control slides were incubated 
with either an IgG fraction of normal rabbit serum at 5 .mu.g/ml (diluted 
in TBS containing 5% swine serum and 0.1% BSA) or without primary 
antibodies. Tissues were then washed in TBS/0.1% BSA and incubated for 60 
min. at room temperature with biotinylated swine anti-rabbit second 
antibody in TBS/0.1% BSA. After washes with this buffer, the sections were 
exposed to avidin-biotin complex for 60 min at room temperature and again 
washed in TBS/0.1% BSA. Slides were reacted with 0.05% diaminobenzidine in 
50 mM Tris-HCl (pH 7.4) with 0.1% hydrogen peroxide for 5 min and 
counterstained in hematoxylin. 
Results 
Western blot analysis showed that each of the three anti-TGF.beta. isoform 
antibodies was specific for its particular isoform and did not react with 
the other two TGF.beta. isoforms. 
Immunohistochemical staining for the three mammalian TGF.beta. isoforms the 
rabbit polyclonal antibodies described above revealed a differential 
expression wounds treated with HA-AP compared to control wounds (See, 
also, Cabrera R. C. et al. (1993) Plast Surg Res Council). 
The results, are summarized in tabular form in FIGS. 1 and 2. Briefly, in 
unwounded sheep skin, TGF.beta.1 was concentrated in the stratum corneum 
of the epidermis, while TGF.beta.2 and TGF.beta.3 were concentrated in the 
strata granulosum, spinosum, and basalis. In the dermis, there was no 
staining with the ant-TGF.beta.1 antibody, whereas anti-TGF.beta.2 stained 
mildly and anti-TGF.beta.3 stained moderately. Merocrine sweat glands 
stained moderately for TGF.beta.1 and TGF.beta.2, but only mildly for 
TGF.beta.3, while sebaceous sweat glands and hair follicles stained 
moderately for TGF.beta.2 and TGF.beta.3 but only mildly for TGF.beta.1. 
Endothelial cells showed little or no immunoreactivity for any of the 
three isoform-specific antibodies. 
Wounded skin, showed a similar distribution of the immunostaining pattern 
in epidermis, dermis, hair follicles, and sebaceous sweat glands. However, 
the staining was more intense relative to unwounded skin through day 14. 
Interestingly, migrating epithelium arising from both the wound margin and 
adjacent hair follicles showed no staining of any TGF.beta. isoform until 
complete reepithelialization by day 7 in the excisional wounds and day 5 
in the incisional wounds. The inflammatory exudate contained a dense band 
of neutrophils and macrophages which showed varying degrees of 
immunoreactivity from none to intense for all three TGF.beta. isoforms. 
Granulation tissue at the exudate-wound interface exhibited intense 
staining for all three isoforms with TGF.beta.3 and TGF.beta.2 being 
greater than TGF.beta.1. Inflammatory exudate separated from underlying 
granulation tissue by migrating epithelium was devoid of staining. By 21 
days, staining for the three TGF.beta. isoforms was similar to that of 
unwounded skin, except for the dermis, in which new scar showed persistent 
immunostaining for all three isoforms, especially TGF.beta.3. Incisional 
wounds exhibited similar staining patterns. 
Lack of staining in the migrating epithelium is consistent with the notion 
that TGF.beta. isoforms influence cell migration during wound repair by 
altering the cells' adhesive properties. Possible mechanisms include 
changes in integrin receptor expression and ECM modification. Increased 
expression of TGF.beta. isoforms continues in the epithelialized dermis to 
day 21. This persistent differential expression may be responsible for the 
excessive ECM and collagen found in scar tissue and the subsequent 
remodeling that occurs as wounds mature. 
The above findings highlight the important role of peptide growth factors 
in the dynamic process. These results are consistent with what is known 
about the isoform-specific effects of TGF.beta. in wound repair and thus, 
not only demonstrate the in vivo biological activity of the HA-AP but also 
suggest a possible mechanism of action. Ellis and coworkers (Ellis I. et 
al. (1992) J Cell Sci 102:447-456) have demonstrated that TGF.beta.1 can 
decrease both hyaluronan production and migration into a collagen gel of 
fibroblasts in vitro. 
Recent unpublished work by Ferguson and coworkers confirms that the 
addition of antibodies to TGF.beta.1 and TGF.beta.2 to adult wounds 
decrease scarring (Shah M. et al., supra). 
The present inventors therefore envision a competitive association where, 
on one side, wound fibrosis and matrix accumulation is enhanced by 
TGF.beta.1 and TGF.beta.2 as they decrease hyaluronan production in the 
ECM thereby exposing hyaluronan associated proteins to proteases in the 
wound fluid. On the other side, collagen and matrix organization is 
improved as hyaluronan protected associated proteins down-regulate 
TGF.beta.1 and TGF.beta.2 and up-regulate TGF.beta.3. 
It is therefore postulated that the hyaluronan of the HA-AP complex 
protects the associated proteins from degradation, and the hyaluronan 
associated peptides effect growth factor expression and collagen 
organization and therefore can be used to modulate scarring. 
EXAMPLE V 
Identification of 62 kDa Hyaluronan-Associated Protein as Calreticulin 
As previously stated, the present inventors isolated and purified HA-AP 
complex from 100-day fetal sheep skin and showed the dominant band on 
SDS-PAGE to be a 62 kDa polypeptide. 
An N-terminal amino acid sequence analysis was performed using the 
electroblot method permitted determination of the first 15 residues. 
Comparison to known protein sequences revealed that calreticulin was the 
only protein with an identical N-terminus. Calreticulin is an 
intra-endoplasmic reticulum low affinity high capacity calcium binding 
protein found in non-skeletal muscle cells which shows remarkable homology 
between mammalian species. 
These results indicate a role for calreticulin in wound healing since it is 
known to bind certain .alpha. subunits of integrins, the cell surface 
proteins mediating many cell-cell and cell-matrix interactions during 
wound repair. 
Despite the fact that calreticulin is thought to be localized in the 
endoplasmic reticulum, a pool of 60 kDa peptides homologous to 
calreticulin was found free in the soluble cytosol, indicating the 
feasibility of an interaction of this peptide with the integrin .alpha. 
subunits (Rojiani M. V. et al. (1991) Biochem 30:9859-9866). 
Furthermore, while calreticulin is immunolocalized to both the endoplasmic 
reticulum and nucleus in proliferating myocytes, the addition of TGF.beta. 
which induces terminal differentiation of the myocytes diminishes the 
intranuclear staining (Opas M. et al. (1991) J Cell Physiol 149:160-171). 
Thus, prior to terminal differentiation calreticulin is immunolocalized 
differently within the cell. 
This behavior reminiscent of the fetal 62 kDa HA-AP which is only present 
early in gestation when fetal skin retains the ability to heal by 
regeneration but is absent by the mid-third trimester when it heals with 
typical post-natal scarring. 
The present inventors propose that these hyaluronan associated proteins, in 
particular calreticulin, play a significant role in the in vivo 
organization of scar tissue. In the fetus, the dominant HA-AP is a 62 kDa 
peptide, calreticulin, which is believed to contribute to the near perfect 
collagen organization seen in early fetal wounds. It is proposed that the 
hyaluronan, although not directly responsible for the biologic effect of 
scarless healing, protects associated proteins from wound proteases. This 
is supported by the finding that selective protease digestion did not 
alter in vitro biological activity of HA-AP unless preceded by 
hyaluronidase treatment. 
Thus, based on the above results and our growing understanding of fetal ECM 
remodeling, the present inventors conceived of the use of calreticulin in 
novel compositions and methods for the ultimate goal of avoiding scarring 
in the wound healing process. 
EXAMPLE VI 
Not All Fetal Tissue Wounds Heal without Scarring 
Bilateral incisional diaphragmatic wounds were created in 100 day gestation 
fetal lambs (term=145 days). The right thoracotomy wound was closed to 
exclude amniotic fluid. In contrast, an Eloesser flap was created at the 
left thoracotomy site, thus permitting the left diaphragmatic wound to be 
continually bathed in amniotic fluid (FIG. 3). 
Wounds were harvested at one, two, seven, or 14 days following wounding and 
analyzed by light microscopy and immunohistochemistry with antibodies to 
collagen types I, III, IV, and VI. 
Whether bathed in or excluded from amniotic fluid, the mesothelial-lined 
diaphragm healed with scar formation and without evidence of muscle 
regeneration. Interestingly, wounds exposed to amniotic fluid were covered 
by a thick fibrous peel of collagen similar to that seen in gastroschisis 
bowel. These findings indicate that not all fetal tissues share the unique 
scarless healing properties of fetal skin. 
The references cited above are all incorporated by reference herein, 
whether specifically incorporated or not. 
Having now fully described this invention, it will be appreciated by those 
skilled in the art that the same can be performed within a wide range of 
equivalent parameters, concentrations, and conditions without departing 
from the spirit and scope of the invention and without undue 
experimentation. 
While this invention has been described in connection with specific 
embodiments thereof, it will be understood that it is capable of further 
modifications. This application is intended to cover any variations, uses, 
or adaptations of the invention following, in general, the principles of 
the invention and including such departures from the present disclosure as 
come within known or customary practice within the art to which the 
invention pertains and as may be applied to the essential features 
hereinbefore set forth as follows in the scope of the appended claims.