WO1994009800A1

WO1994009800A1 - A method of promoting wound healing and compositions useful for same

Info

Publication number: WO1994009800A1
Application number: PCT/AU1993/000554
Authority: WO
Inventors: Mark Werner Schulz; Peter James Davey
Original assignee: Granulon Pty. Limited
Priority date: 1992-10-28
Filing date: 1993-10-27
Publication date: 1994-05-11
Also published as: CA2147508A1; EP0669828A4; EP0669828A1; JPH08502496A; ZA938054B; AU5365894A

Abstract

The present invention relates to a method of inducing, stimulating, enhancing, accelerating or otherwise promoting wound healing by the application of vitreous from mammalian ocular tissue or an extract or one or more components of the vitreous. The present invention is also directed to a topical composition comprising vitreous or an extract or one or more isolated components thereof capable of differentially stimulating growth and proliferation of granulation tissue but with substantially no effect on skin tissue.

Description

A METHOD OF PROMOTING WOUND HEALING AND COMPOSITIONS USEFUL FOR SAME

The present invention relates to a method of inducing, stimulating, enhancin accelerating or otherwise promoting wound healing by the application of vitreo from mammalian ocular tissue or an extract or one or more components of th vitreous. The present invention is also directed to a topical composition comprisin vitreous or an extract or one or more isolated components thereof capable differentially stimulating growth and proliferation of granulation tissue but wit substantially no effect on skin tissue.

The term "vitreous" is used in the subject specification as a short hand notation f the 'Vitreous humour" or "vitreous body" which adopts the nomenclature propose by Balazs and Denlinger (1984). The vitreous is considered to include th connective tissue surrounded by the lens, ciliary body and retina.

For the purposes of describing the present invention, the skin is considered to consi of an outer epidermal layer and an inner dermal layer. These two layers rest on third subcutaneous tissue layer; from which granulation tissue is derived. Reference herein to "skin tissue" is taken as reference to the epidermal and derm layers but not the subcutaneous /granulation.

There are three overlapping phases of tissue response to injury: inflammatio granulation tissue production; and matrix formation and remodelling. These phas are shown in Figure 1.

The inflammatory phase is associated with disruption of blood vessels. This resul in extravasation of blood cells and plasma components into surroimding tissue an the clotting process which also involves platelet release of chemoattractants an mitogens. This in turn leads to further invasion of the wounded tissue by blood cel such as neutrophils and monocytes which, like platelets, are crucial to the next phas of wound healing (Clarke, 1988).

Granulation tissue consists of a mixture of fibroblasts, monocyte-derived and tissu macrophages and new blood vessels. The term "granulation tissue" derives from i granular appearance which is histologically due to the numerous newly formed bloo vessels. Granulation tissue may be found at sites of wound repair such as in chron non-healing wounds or at sites of bone fracture where the granulation tissue composed largely of specialised myofibroblast referred to hereinafter as "osteoblasts Proliferation of fibroblasts and the growth of new blood vessels into the wound occ simultaneously and interdependently and are stimulated by chemoattractants an growth factors from macrophages and platelets. Such chemoattractants and growt factors include thrombin, fibroblast growth factor (FGF), epidermal growth fact (EGF) and platelet-derived growth factor (PDGF) (Leibovich and Ross, 1975). R vascularization occurs from capillaries sprouting from adj acent blood vessels followe later by smooth muscle recruitment. This stage is an important part of wound repa since it supplies oxygen and essential nutrients to the proliferating repair tissue.

In deep wounds lacking skin, fibroblasts migrate into the wound space from th subcutaneous layer and lay down large amounts of loose extracellular matrix, initial rich in fibronectin and hyaluronic acid (Kurkinen et al., 1980). Within the woun itself, the fibroblasts undergo a phenotypic modulation to become the so-calle "myofibroblasts". The myofibroblasts have an increased mobility and contractil capacity without losing matrix synthetic function. Myofibroblasts are the mo numerous cells in granulation tissue. They are actin-rich and become aligned withi the wound and their contraction is responsible for wound closure (Gabbiani et a 1972). Re-epithelialkation occurs concurrently with fibroplasia and is an importa mechanism to prevent bacterial infection and loss of tissue fluid. The epithelial cel undergo marked phenotypic alteration concomitant with migration that result i increased cell mobility. Like many other cell types, migration of epithelial cells do not depend on cell proliferation. Once re-epithelialization is complete, the cel revert to their original phenotype.

The third overlapping phase of wound repair is matrix formation and remodellin The composition and structure of the extracellular matrix of granulation tissu changes continuously from the time it is first deposited and is controlled by cytokin such as transforming growth factor- β [TGF-β] (Roberts et al, 1986). During th formation of granulation tissue, fibronectin provides a substratum for the migratio of cells, a linkage for myofibroblasts to effect wound contraction, and adhesion sit for collagen fibril formation. Collagen types I, III and V appear and provide tensil strength for the wound and proteoglycans provide resilience to deformation of th tissue.

Wounds requiring healing by secondary intention are wounds with edges which ar widely separated such that no epidermis or dermis is present. As a consequenc healing progresses from the base upwards as well as from the edge inwards. Th dermis shows little wound healing activity in this respect and the majority granulation tissue which eventually fills the wound site comes from the subcutaneo fat at the wound edge and from the subcutaneous floor of the wound. As note above, granulation tissue is formed by the proliferation and migration of th surrounding connective tissue element of the subcutaneous tissue; it is composed i the first instance of capillary loops and fibroblasts together with a variable numb of inflammatory cells.

The clinical need to provide prompt, functional coverage and to prevent sc formation in deep, extensive secondary indication wounds and the need to initiat and accelerate healing in chronic non-healing secondary indication wounds, has le to the experimental use of a vast number of materials in human trials.

These materials are generally ill-defined combinations of growth factors, which hav been derived from a number of sources (see Van Brunt and Klausner, 1988; Buckle et al. 1985).

Platelet Derived Wound Healing Formula (PDWHF) has been used, topically, in a attempt to accelerate wound healing. It is considered likely that PDWHF stimulat repair of chronically non-healing cutaneous wounds by a combination of growt factors present in the formula. One study showed that wounds of different etiologi responded equally well to PDWHF (Knighton et al- 1988).

In an extensive study in human subjects, Carter et al. (1988) used three crud preparations: autologous serum, porcine lysate (obtained from porcine keratinocyte and bovine platelet lysate. All three showed limited success in trials on patients wit non-healing, deep-ulcerated wounds (Carter et al, 1988).

Extracts from mammalian eyes have also studied for their potential wound heali properties. In GB 1 342761, a whole eye extract was shown to enhance cicatrisatio of corneal wounds. Furthermore, GB 1 603 034 discloses an aqueous solution of tissue regenerating extract such as from vitreous which assisted regeneration of cel of the epidermal layer. A similar effect on epidermal cells was disclosed in U Patent No. 4,670,257 using an aqueous salt extract of ocular tissue, such as fro vitreous.

There is a need to identify and develop substances capable of accelerating woun healing by selectively targeting granulation tissue but not cells of the epidermal an dermal layers of the skin. Such substances will provide a major advance in woun healing management especially for secondary intention wounds which lack epiderm and dermis. These substances would also be useful for promoting granulation tiss during repair of fractured bones. Accordingly, one aspect of the present invention provides a vitreous formulatio from mammalian ocular tissue which is capable of preferentially accelerating growt of granulation tissue compared to skin tissue. The vitreous formulation m comprise isolated vitreous, vitreous extract or one or more components of a vitreo having the desired activity. A vitreous extract may be an aqueous extract or a sa extracted preparation.

The vitreous formulation is generally adapted or in a form suitable for topic administration. According to this aspect of the present invention there is provide a topical composition comprising vitreous or an extract thereof from mammalia ocular tissue wherein said vitreous or its extract preferentially accelerates growth granulation tissue compared to epidermal and dermal tissue.

For convenience reference to an "extract" of vitreous includes reference to one more active components isolated and/or purified therefrom. Furthermore, by w of shorthand notation, the vitreous formulations and topical compositions of th present invention are hereinafter referred to as a vitreous based healing formulatio (VBHF). The term "topical composition" is used herein in its broadest sense an includes the application of the composition to one or more layers of subcutaneo tissue and directly at the site of bone fracture.

The expression "epidermal tissue" is used in its broadest sense and is particular directed to keratinocytes. "Dermal tissue" is also used in its broadest sense an includes skin fibroblasts. Sub-cutaneous /granulation tissue is also used in i broadest sense and includes vascular smooth muscle cells and myofibroblasts. T present invention is predicated in part on the surprising discovery by the invento that the VBHF stimulates proliferation of vascular smooth muscle and myofibroblas but substantially not skin fibroblasts and keratinocytes. According to a preferred aspect of the present invention, there is provided a topic composition comprising vitreous or an extract thereof from mammalian ocular tissu wherein said vitreous or its extract preferentially stimulates growth of vascula smooth muscle cells and/or myofibroblasts while substantially not stimulating growt of skin fibroblasts and/or keratinocytes. The preferential effect is conveniently an particularily observed and monitored in vitro.

Reference herein to a "vitreous extract" includes vitreous isolated away from th mammalian ocular tissue, vitreous following at least one step of purification o fractionation or any active components therein. The mammal is generally a livestoc animal such as a bovine, ovine, equine or porcine animal or a goat or a laborator test animal such as a murine animal, rabbit or guinea pig. The most preferre source of the vitreous of the present invention is from a bovine animal.

In another embodiment, the vitreous is fractionated and one or more component or a mixture of components isolated and purified and which have a simila differential effect on granulation and skin tissue as does the vitreous or extrac thereof.

According to this aspect of the present invention, there is provided a componen isolateable from mammalian vitreous or extract thereof, said component capable o accelerating or promoting growth of granulation tissue such as vascular smoot muscle cells or myofibroblasts while having a minimal proliferative effect on ski cells such as keratinocytes and dermal derived fibroblasts. Preferably the compone is in isolated and purified form meaning that a composition of matter comprises least 20%, more preferably at least 35%, even more preferably at least 45%, sti more preferably at least 55-65% and even still more preferably at least 75-90% o the component as determined by weight, activity, antibody binding or othe convenient means. The VBHF of the present invention may further comprise one or more exogenou factors added to the composition and which may also assist the wound healin process. Such exogenous factors contemplated in this aspect of the present inventio include but are not limited to PDWHF, FGF, EGF or insulin-like growth facto (IGF), one or more antibiotics or other antimicrobial agents or vitamins such a vitamin E, D and/ or K. The VBHF may also comprise one or mor pharmaceutically acceptable carriers and/or diluents.

The VBHF may be topically applied directly or after incorporation into a medicate foam, gel, cream or liquid. Alternatively, it may be incorporated into a solid matri such as a bandage, dressing, gauze or sutures. The VBHF maybe in isolated and/o purified form or may be frozen or lyophilized, in which case it is reconstituted prio to use. Reference can conveniently be made to the British Pharmacopoeia (1980) for description of conventional agents which may be usefully incorporated into VBH

The VBHF of the present invention is contemplated to be particularly useful i wound healing by secondary intention of wounds which require accelerated growt of granulation tissue such as ulcers which may be associated with diabetes, peripher vascular disease or pressure sores or bed sores; dermal tissue damage such a following various degrees of burns; post operative skin lesions lacking skin wher accelerated growth of subcutaneous/granulation tissue is required; accelerate healing of granulation tissue in bone fractures; accelerated growth o subcutaneous /granulation tissue prior to application of a skin graft.

According to this aspect of the present invention there is contemplated a method o stimulating, enhancing, accelerating or otherwise promoting healing of a wound o an animal, said method comprising contacting said wound with an effective amou of VBHF as hereinbefore defined for a time and under conditions sufficient fo wound healing to occur. Such wounds -are generally healed by secondary intention as defined above. T effective amount is the amount of VBHF effective to stimulate, enhance, accelera or otherwise promote wound healing which is conveniently assessed by stimulati of vascular smooth muscle cells or myofibroblasts with substantially no stimulati effect on keratinocytes or skin fibroblasts. Reference above to a "wound" includ reference to a bone fracture.

The animal to be treated may be a human, livestock animal as hereinbefore define a laboratory test animal as hereinbefore defined, a companion animal (e.g. dog cat) or a captive wild animal. Most preferably, however, the animal is a human.

The wound healing protocol may comprise a single application of VBHF or a regi involving multiple applications of VBHF on an hourly, twice daily, daily, multip daily, weekly, multiple weekly or monthly basis, depending on the wound to treated, the extent of the wound and the healing capacity of the patient. T method may also comprise further treatment with one or more other agents such antibiotics or other antimicrobial agents, anaesthetics or other wound heali promotents. VBHF may be used as isolated, or may require concentration dilution. Dilutions contemplated herein are in the order of 1:2 to about 1: although dilutions of the order 1:2 to 1:5 are preferred. If the VBHF is concentrat this may be done by any number of procedures including lyophilisation or filtrati and extend to a concentration factor of 2 to 10.

In another embodiment, the VBHF is used in a non-liquid form such as followi lyophilisation. Such a form may be particulary useful for the application to a sol matrix such as a bandage, dressing, gauze or sutures. The present invention is further described by reference to the following non-limitin Figures and/or Examples.

In the Figures:

Figure 1 is a graphical representation showing the phases of wound repair.

Figure 2 is a graphical representation of the effect of VBHF on tritiated - thymidine incorporation into vascular smooth muscle cells.

Figure 3 is a graphical representation showing the effect of VBHF on tritiated - thymidine incorporation into myofibroblasts.

Figure 4 is a graphical representation showing the effect of VBHF on tritiate thymidine incorporation into osteoblasts.

Figure 5 is a graphical representation of the effect of VBHF on tritiated - thymidine incorporation into skin fibroblasts.

Figure 6 is a graphical representation showing the effect of VBHF on tritiated - thymidine incorporation into keratinocytes.

Figure 7 is a graphical representation of the effect of VBHF on tritiated - thymidin incorporation into vascular endothelium.

Figure 8 is a graphical representation showing the effect of VBHF on proliferatio of vascular smooth muscle cells.

Figure 9 is a graphical representation showing the effect of VBHF on proliferatio of myofibroblasts. Figure 10 is a graphical representation showing the effect of VBHF on proliferatio of skin fibroblasts.

Figure 11 is a graphical representation showing the effect of VBHF on proliferatio of keratinocytes.

Figure 12 is a graphical representation showing the effect of VBHF on proliferatio of vascular endothelium.

Figure 13 is a graphical representation showing the effect of VBHF on migratio of vascular smooth muscle cells.

Figure 14 is a graphical representation showing the effect of VBHF on migratio of myofibroblasts.

Figure 15 is a graphical representation showing the effect of VBHF on migratio of skin fibroblasts.

Figure 16 is a graphical representation showing the effect of VBHF on migratio of keratinocytes.

Figure 17 is a photographic representation showing the effect of VBHF o morphology of vascular smooth muscle cells. In the presence of: A DMEM to 0.5 v/v FCS; B. DMEM to 0.5% v/v FCS and 1:2 diluted VBHF; C. DMEM to 0.5 v/v FCS to 1:4 diluted VBHF.

Figure 18 is a photographic representation showing the effect of VBHF o morphology of myofibroblasts. In the presence of: A DMEM to 0.5% v/v FCS; DMEM to 0.5% v/v FCS and 1:2 diluted VBHF; C. DMEM to 0.5% v/v FCS to 1: diluted VBHF. Figure 19 is a photographic representation showing the effect of VBHF o morphology of skin fibroblasts. In the presence of: A DMEM to 0.5% v/v FC B. DMEM to 0.5% v/v FCS and 1:2 diluted VBHF; C. DMEM to 0.5% v/v FCS t 1:4 diluted VBHF.

Figure 20 is a photographic representation showing the effect of VBHF o morphology of keratinocytes. In the presence of: A DMEM to 0.5% v/v FCS; DMEM to 0.5% v/v FCS and 1:2 diluted VBHF; C. DMEM to 0.5% v/v FCS to 1: diluted VBHF.

EXAMPLE 1

Isolation of vitreous

The -vitreous is removed from a freshly slaughtered bovine's eye in the followin manner. Preferably, all procedures outlined below are carried out in a laminar flo hood.

(i) the outside of the eye is rinsed in 70% v/v ethanol. (ii) a syringe fitted with a 19G needle is inserted into the cavity containin the aqueous humour and the aqueous humour is collected, (iii) an incision is made around the cornea with a scalpel and the corne is removed, (iv) using a pair of forceps, the lens is removed. (v) the vitreous is then collected in a 10ml sterile syringe.

(vi) the syringe and its contents are then frozen at -70 degrees Celsius. EXAMPLE 2 Preparation of vitreous based healing formulation

The VBHF is used as isolated according to the method of Example 1 or addition components added such as one or more pharmaceutically acceptable carriers and/ diluents, emulsifying components such as Beclomethasone or Cetrimide components selected from saline, protein, antibiotic, carbohydrates. Alternativel or in addition to, the vitreous is fractionated to remove unwanted components or t isolate and purify desired active components.

Once the VBHF is prepared, it is then stored or transported as is or in a frozen lyophilised condition.

EXAMPLE 3

Wound Treatment

(i) The wound is debrided and if infection is present it is removed by t application of a suitable antibiotic or antimicrobial agent.

(ii) The wound is washed in 0.9% w/v sterile saline.

(iii) An aliquot of 1ml of the VBHF is applied from a syringe and disperse evenly over the wound.

(iv) A piece of plastic backed gauze (cut to the same size of the wound) moistened with 0.9% w/v sterile saline and laid over the wound.

(v) Plastic wrap is placed over the gauze.

(vi) Treatment is repeated every 12hrs. EXAMPLE 4

Preparation for Skin Grafting

(i) Steps (i) to (v) of Example 3 are carried out until a suitable bed o granulation tissue is present.

(ii) A skin graft can then be applied to the growing dermal tissue. 2

(iii) Treatment with VBHF can be continued if required.

EXAMPLE 5 Treatment of Ischaemic Ulcer

A clinical trial was carried out in a hospital environment. A patient was a male 7 years of age in good health with the exception of a 5cm x 3cm ulcer on the lower le leg. The ulcer had failed to respond to various proprietary dressings which had bee applied over a period of six months. In addition, during this treatment period, ski grafting had also been carried out and as a result, the patient required frequen hospitalisation. All of these treatment regimes were unsuccessful. The woun lacked epidermal and dermal tissue, secondary intention healing by extensiv granulation tissue growth was required.

The patient was started on a treatment regime which involved an application, ever 12 hours, of the VBHF, as outlined in Example 3. As a result of this treatme regime, the patient's ulcer healed completely in 14 days with no recurrence for th period of the test which was 18 months. EXAMPLE 6 Treatment of Diabetic Ulcer

A clinical test was carried out in a hospital environment. A patient was a male 7 years of age in good health with the exception of* a chronic ulcer measuring 7cm 4cm on lower right leg. The ulcer had been recurrent over 25 years during whic time healing had never been complete using various proprietary dressings and ski grafting techniques. Accelerated growth of granulation tissue was required t successfully heal the wound.

The patient was commenced on a treatment regime using a topical application of th VBHF as outlined in Example 2. As a result of this treatment regime, the patient' ulcer was 90% healed after 12 days and completely healed after 22 days, with n recurrence of the ulcer for the time of the test which was 17 months.

EXAMPLE 7

Treatment of Post-traumatic Ulcer

A clinical trial was carried out initially in a hospital setting and later by the patie at home. The patient was a female 48 years of age with a post traumatic no healing ulcer.

The ulcer measured 5cm x 5cm and was situated on the lower left le No progress had been made in healing the ulcer using various dressing techniqu and there was no evidence of new skin growth. In addition, over the 5 year histor of the ulcer, repeated skin grafting resulted in short term coverage of the wound, b each skin graft eventually degenerated. The wound lacked epidermis tissue includin both epitheial and dermal tissue and required granulation tissue growth for successf wound healing. The patient was commenced on a treatment regime which involved the topic application of the VBHF as outlined in Example 3. As a result of this treatment, t ulcer was completely healed after 21 days.

EXAMPLE 8 Treatment of Diabetic Ulcer

A clinical trial was carried out in a hospital setting on a female 82 year old patie The patient had 2 ulcers: one on the front of the right foot (5cm x 2cm) and t other at the rear of the right leg (10cm x 10cm). Both ulcers were the result peripheral vascular restriction resulting from diabetes. The ulcers had failed respond to various proprietary dressings and hyperbaric treatment over 5 mont The ulcer at the rear had penetrated through the tendon and amputation of the li was likely.

The patient was commenced on a treatment regime for the ulcer on the back of t right leg which involved the topical application of the VBHF (as outlined in Examp 3). The ulcer on the front of the right foot was dressed with N-saline as a contr After 14 days the ulcer on the back of the right leg had extensive growth granulation tissue, whereas the ulcer on the front of the right foot showed no chang Treatment of the ulcer on the rear of the right leg was then commenced as outlin in Example 4. In both cases rapid growth of granulation tissue occurred, the ulc at die front of the right foot was completely healed after 22 days whereas the ulc on the front of the right foot was completely healed after 38 days. EXAMPLE 9 Differential effect of VBHF

1. Sources of Cells

Vascular Smooth Muscle Cells

Vascular smooth muscle cells were obtained by enzymatic dispersion of 12 week ol rabbit aortae by standard procedures (Campbell and Campbell, 1993). Cells wer grown in Dulbecco's Minimal Essential Medium plus 0.5% v/v foetal calf seru (DMEM + 0.5% v/v FCS).

Myofibroblasts

Myofibroblasts were obtained by enzyme dispersion of Selye's granuloma pouc induced in rats by subcutaneous injection of 1 ml of 1% v/v croton oil in corn oi following a 20 ml bolus of air (Selye, 1953). Cells were grown in DMEM + 0.5 v/v FCS.

Skin Fibroblasts

Human skin fibroblasts were obtained from Dr Rebecca Mason of the University o Sydney, New South Wales, Australia and were grown in DMEM + 0.5% v/v FC

Keratinocytes

Human keratinocytes (epidermis) were obtained from Dr Rebecca Mason, Universit of Sydney, New South Wales, Australia together with keratinocyte growth mediu (KGM). For experiments, cells were grown in DMEM.

Endothelial Cells

Endothelial cells were isolated from bovine aorta by the method of Horrigan et (1988) grown in medium RPMI + 0.5% v/v FCS. 2. VBHF

VBHF was prepared according to Examples 1 and 2 in plastic syringes on ice.

The VBHF was stored frozen until required, then serially diluted at the time of u at 1:2, 1:4, 1:8 and 1:16 in either DMEM + 0.5% v/v FCS or RPMI + 0.5% v FCS (for endotiielial cells). Penicillin G was used to control contamination.

3. Incorporation of ^■Η-Ηiymidine

Vascular Smooth Muscle Cells Cells were seeded at 5 x 10" cells/well into a 24 well plant in DMEM + 10% v FCS. The cells were allowed to attach and flatten for 18 hours, then the cel thoroughly washed in DMEM + 0.5% v/v FCS. The wells were then divided in six groups, four wells per group and exposed to the following conditions:

1. DMEM + 0.5% v/v FCS

2. DMEM + 0.5% v/v FCS + 1:2 VBHF

3. DMEM + 0.5% v/v FCS + 1:4 VBHF

4. DMEM + 0.5% v/v FCS + 1:8 VBHF

5. DMEM + 0.5% v/v FCS + 1:16 VBHF

The wells containing DMEM + 0.5% v/v FCS acted as controls for those with add VBHF.

The cells were further incubated at 37 °C for 24 hours, at which time 0.5 mCi ³ thymidine (Amersham) was added to each well. After 4 hours incubation, the cel were harvested onto 2.5 cm glass microfibre filters (Whatman International, U washed and then lysed with 3 x 3 ml H₂O. The filters were air-dried, then placed scintillation vials to which 7 ml Beckman Ready Safe scintillant was added. Sampl were counted in a Beckman LS6000TA β-Counter against a blank.

The quadruplicate wells in each experimental condition were meaned and compar statistically to the control (DMEM + 0.5% v/v FCS). A Sigmastat statistic program was utilized for a one way analysis of variance.

It was found that VBHF at a dilution of 1:2 and 1:4 stimulated the ³H-thymidi uptake into smooth muscle cells by about 6.4 fold above controls in each of the experiments (Figure 2). Dilutions of 1:8 and 1:16 also stimulated ³H-thymidi uptake but to a lesser extent and dose dependently. Stimulation of ³H-thymidi uptake was significant for all dilutions tested. VBHF at dilution 1:2 and 1 stimulated ³H-thymidine uptake above that of the positive control (DMEM + 10 v/v FCS) (Figure 2).

Myofibroblasts

Cells were seeded at 5 x 10⁴ cells /well. The protocol was identical to that describ above.

VBHF at 1:2 dilution significantly stimulated ³H-thymidine uptake in myofibroblasts above controls (1.7 fold) in each of the three experiments (Figure There was no significant increase in ³H-thymidine incorporation with any of the oth dilutions (1:4, 1:8 and 1:16).

Skin fibroblasts

VBHF at 1:4 and 1:8 dilutions significantly decreased the incorporation of ³ thymidine into skin fibroblasts while 1:2 and 1:16 had no effect (Figure 5).

Keratinocytes

The protocol was identical to that described above.

VBHF at 1:4, 1:8 and 1:16 dilution decreased incorporation while 1:2 dilution h no effect (Figure 6). Endothelial Cells

The protocol was as described above except that the control medium was RPMI

0.5% v/v FCS.

VBHF at all dilutions induced a significant decrease in incorporation of ³ thymidine into endothelial cells (Figure 7).

4. Cell Proliferation

Experiments were carried out to determine the effect of VBHF at dilutions 1:2, 1: 1:8 and 1:16 on cell proliferation. Cells were plated out and left to attach an flatten for 18 hours. Quadruplicate wells were then counted using haemocytometer, in order to determine number of cells at experimental time 0. Th remaining wells were washed with DMEM + 0.5% v/v FCS (or RPMI + 0.5% v/ FCS for endothelial cells) then fed with their appropriate medium as follows:

1. DMEM + 0.5% v/v FCS

2. DMEM + 0.5% v/v FCS + 1:2 VBHF

3. DMEM + 0.5% v/v FCS + 1:4 VBHF

4. DMEM + 0.5% v/v FCS + 1:8 VBHF 5. DMEM + 0.5% v/v FCS + 1:16 VBHF

For endothelial cells, the protocol for 1 to 5 above was repeated using RPMI 0.5% v/v FCS.

An experiment for each cell type was performed with quadruplicate wells pe condition. Cells were counted at day 2 for vascular smooth muscle cells an myofibroblasts, day 3 for skin fibroblasts and endothelial cells and day 5 fo keratinocytes. This reflected the different rates of proliferation of the cell types.

Vascular Smooth Muscle Cells

The number of smooth muscle cells was significantly increased in the presence of 1: and 1:4 diluted VBHF by 2.5 fold, with no effect on 1:8 and 1:16 (Figure 8). Myofihrohlasts

The number of myofibroblasts was significantly increased above control by VBH diluted at 1:2 only (Figure 9).

Skin Fibroblasts

There was no significant change in number of skin fibroblasts with any dilution VBHF (Figure 10).

Keratinocytes Similarly, there was no significant change in number of keratinocytes with a dilution of VBHF (Figure 11).

Endothelial Cells

VBHF diluted at 1:2, 1:4 and 1:8 was growth inhibitory for endothelial cells (Figur 12). The number of cells at day 0 was 4.08 ± 0.51 x 10⁴ cells/well, while at day with 1:2 diluted VBHF there were 6.78 ± 0.45 x 10⁴ cells per well, and in RPMI 0.5% v/v serum alone at day 3 there were 25.09 ± 3.19 x 10⁴ cells /well. The grow inhibitory effect was dose dependent and cell number returned to the level of contr RPMI + 0.5% v/v FCS at dilution of VBHF of 1:16 (Figure 12).

5. Cell Migration All five cell types were suspended in DMEM + 10% v/v FCS (RMPI + 15% v/ FCS for endothelial cells) at 1 x 10⁵ cells/ml, then 200 μl pipetted into a circul well insert placed in the centre of each well. The cells were allowed to attach an flatten into a confluent layer overnight, the insert removed, the cells thorough washed and the experimental medium added.

For each cell type, quadruplicate wells were used for each experimental conditio The experimental conditions were the same as that described above (1 to 5).

Once the insert was removed and the experimental medium added the cells we allowed to migrate in a radial manner from the central circle of cells over 7 day Fresh medium and VBHF were added on day 3. On day 7, cells were fixed in 10 v/v neutral buffered formalin and stained with 1% v/v toluidine blue. The area o culture substrate covered by cells was determined by point counting morphometr and expressed as a percentage of the total well surface. Quadruplicate wells for eac cell type were fixed and stained at experimental time 0. s

Vascular Smooth Muscle Cells

In the presence of 1:2 and 1:4 dilutions of VBHF vascular smooth muscle cel migration was significantly increased above control wells containing DMEM + 0.5 v/v FCS (Figure 13). The effect was dose dependent.

It should be noted that vascular smooth muscle cells do not grow as a monόlayer and in situations where cell growth is stimulated, the cells can grow in multipl layers. This was especially evident in the presence of 1:2 and 1:4 diluted VBHF an the positive control, where staining with toluidine blue was not only more extensiv over the area of the culture substrate but was markedly more intense.

Myofibroblasts

Only the dilution of 1 :2 VBHF significantly stimulated myofibroblast migration abov control (Figure 14).

Skin Fibroblasts

There was no significant difference in degree of skin fibroblast outgrowth with an dilution of VBHF compared with control (Figure 15).

Keratinocytes

Similarly, there was no effect on keratinocyte migration by any dilution of VBH

(Figure 16).

Endothelial Cells

VBHF at 1:2 and 1:4 dilutions appeared to be toxic for endothelial cells, since th area of culture substrate covered by cells after 7 days was less than that at day prior to exposure to VBHF. The effect was dose dependent, with less toxic effe occurring at 1:8 and 1:16 dilutions.

6. Cell Morphology In order to observe any change in cell shape, size or orientation induced by VBH quadruplicate wells of each cell type were exposed to:

1. DMEM + 0.5% v/v FCS

2. DMEM + 0.5% v/v FCS + 1:2 diluted VBHF 3. DMEM + 0.5% v/v FCS + 1:4 diluted VBHF

For Endothelial cells, 1 to 3 were repeated with RPMI + 0.5% v/v FCS.

Vascular Smooth Muscle Cells In the presence of both 1:2 and 1:4 diluted VBHF, the number of smooth musc cells was clearly greater than in control (DMEM + 0.5% FCS), but there was litt change in cell shape, size or orientation (Figure 17), except that the cells we confluent.

Myofibroblasts

In contrast, myofibroblasts changed shape from flat, broad cells in control (Figu

18A), to an elongated ribbon shape characteristic of motile cells in the presence

1:2 diluted VBHF (Figure 18B). This was also the case, although to a lesser exte with 1:4 dilution VBHF (Figure 18C).

Skin Fibroblasts

Skin fibroblast morphology did not alter appreciably in the presence of 1:2 or 1 diluted VBHF or 0.5% FCS (Figure 19).

Keratinocytes

When the keratinocytes were first passaged, the cells were flat, epidermal-like a growing in a monolayer. Two distinct cell populations were evident, with a small population of larger cells spread uniformly throughout the monolayer. A proliferation and migration studies were carried out with the cells in this phenotyp and 1:2 and 1:4 dilutions of VBHF had no effect on morphology. The results ar shown in Figure 20.

EXAMPLE 10 Proliferative Effect of VBHF on Osteoblasts

Cultures of human foetal osteoblast-like cells were treated with VBHF an compared to the effects produced by vitamin D l,25(OH)₂D₃ and TGF-β, both which modulate the rate of cell division of osteoblast-like cells in vitra

Parameters tested included comparison of cell numbers and [³H]- thymidin incorporation into the cells after 48 hours treatment. The known and unknow treatments were tested against two control treatments. The first was a culture whic had received no treatment and the second set of controls included cells cultivate in medium that had been diluted to 75% w/v of its initial concentration usin Dulbecco's PBS.

Compositions were received on ice and stored in a 0-4 °C refrigerator.

Two flasks of the primary cell culture FBC200893 (from the trabecular ends of lon bones) were trypsinised and the cells were washed and resuspended at concentration of 6 x 10⁴ cells/ml of BGJ medium (GIBCO), supplemented wit phosphoascorbate and 10% v/v FCS. The cells were plated in one ml aliquots in 2 well culture plates overnight.

The medium was aspired the following day and replaced and 1 ml/well 2% v/v FC supplemented BGJ medium. The methods by which the various factors wer delivered to the cultures are outlined in Table 1. Treatments, performed i quadruplicate, were randomly allocated to the columns of the 24 well plates. TABLE 1 Treatments used in the study and the composition of those treatments

Treatments Used in Study Composition

PBS control PBS:BGJ 1:3

TGF- β 0.5 ul of 1 μg/ml TGF- β

VBHF VBHRBGJ 1:3

Nil Control 2% FCS in BGJ*

10^"8 l,25(OH)₂D₃ lμl 10^"5M 1,25D dissolved in the E OH

* All treatment cultures used this formulation as the basis of the treatme medium.

The cultures with various treatments were incubated for 48 hours after whi cultures were used to measure cell density or [³H]-thymidine incorporation.

Variations in the cell cultures as a result of treatment were tested using ANOV from the Microsoft Excel package. Significance of differences between treatmen was performed using Tukey's method of multiple comparison at p<0.05.

Treatment of the cells with TGF-β at a concentration of 0.5 ng/ml affected t morphology of the cells. Cultures appeared more confluent than those treated wi either PBS or Nil treatment. Cultures treated with TGF- β appeared to be compos of a higher percentage of cells with cuboidal morphology while PBS control cultur were dominated by cells expressing a fibroblastic morphology.

Cultures treated with VBHF exhibited similar morphology and culture surfa coverage as cultures treated with TGF-β. Cultures exposed to these two treatmen appeared to be fully confluent (totally covering the culture surface). TABLE 2 Cell Counts

Using ANOVA at the 95% confidence interval, there is a statistically significan difference in cell number between cultures treated with VBHF and TGF- β and PB treated cultures.

TABLE 3 [^-Thymidine Incorporation

The results of ³H-thymidine incorporation are shown in Figure 4. Analysing these data using ANOVA shows there is no significant difference in t rate of thymidine uptake between cultures maintained in untreated 2% FC supplemented BGJ medium and cultures in which the medium has been diluted t 75% of its original concentration with Dulbecco's PBS. There is a significa difference in the rate of thymidine uptake between cultures treated with PBS an those treated with VBHF (p<0.05).

Dilution of the culture medium to 75% of its original concentration using PB appears to have had no significant effect on the cell numbers or the rate of ce division of the human foetal bone derived cells compared to the untreated contr cultures. This was, therefore, considered to be more appropriate as a control for th purpose of testing the effect of the supplied composition, as the use of thes compositions entailed diluting the culture medium to 75% of its norm concentration.

TGF- β has been shown to have a number of effects on bone cells, depending on th system in which the factor is used. In the present study, TGF-β decreased ce proliferation and there is a corresponding decrease in thymidine uptake. Th inhibition of cell division by TGF-β treatment was probably related to the ce density used in this study. At low cell densities, osteoblast-like cell cultures tend t be dominated by cells which are at an early stage of differentiation, an correspondingly, a stage of rapid cell division. In such cases the predominant effe of TGF- β is to cause the cells to advance their stage of differentiation simultaneous decreasing the rate of cell division. In contrast, when bone cell cultures are dense the cells tend to differentiate. This leads to a decrease in the rate of cell divisio When such cultures are treated with TGF-β they have a tendency to increase t rate of cell division. This accounts for the different effects of TGF-β on the rate cell division under different conditions. The state of differentiation in the present study can be assessed qualitatively b observation of the cell morphology. Differentiated osteoblasts in culture tend t exhibit a more cuboidal morphology while cells that are less well differentiate appear more like cultured fibroblastic cells. TGF-β and VBHF appeared to affec the morphology of the cells in culture as a greater percentage of the cells in the culture were cuboidal in shape. More qualitative methods of assessing state o differentiation can be made by measuring biochemical markers of differentiatio such as production of alkaline phosphatase.

Cells treated with VBHF were more cuboidal and larger than cells of control cultures. Further, cultures treated with this composition were completely confluent at the end of 48 hours. Treatment of the bone cell cultures with VBHF appeare to significantly reduced cell number compared with control cultures over the 48 hou period of incubation. There was, however, a stimulation of thymidine incorporatio at the end of the 48 hour period.

The apparent discrepancy between cell numbers at the end of 48 hours (a measure of proliferation during the period of treatment) and thymidine incorporatio measured over 4 hours at the end of the 48 hour treatment, may reflect the activit of the cells at two different time periods. It is likely that VBHF has a differentiatin effect. The differentiation of the cells in these cultures may be followed by a increase in the proliferative activity as measured by thymidine incorporation. This increase in proliferation may not be measurable in terms of cell numbers for som period of time beyond the initial 48 hour period. REFERENCES:

Knighton, D., Doucette, M., Fiegel, V., Ciresi, K., Butler, E. and Austin, L. (1988) The Use of Platelet Derived Wound Healing Formula in Human Clinical Trial, in p.p. 319-329.

Carter, D., Balin, A, Gottlieb, M., Eisinger, A, Lin, A, Pratt, L., Sherbany, A and Caldwell, D. (1988) Clinical Experience with Crude Preparations of Growth Factors in Healing of Chronic Wounds in Human Subjects, in p.p. 303-317.

Van Brunt, J. and Klauser, A (1988) Growth Factor Speed Wound Healing. Biotechnology, 6. p.p. 25-30.

Buckley, A, Davidson, J., Kamerath, C, Wolt, T. and Woodward, S. (1985) Sustained Release of Epidermal Growth Factor Accelerates Wound Repair. Proc Natl Acad Sci USA, 92, p.p. 7340-7344.

Balazs, E. and Denlinger, J. (1984) The Vitreus in Dawson, H. The eye (3rd Ed) Vol. la Vegetative Physiology and Biochemistry Academic Press, NY., p.p. 533-580.

Clark, RAF. (1988). Overview and general considerations of wound repair. In "The Molecular and Cellular Biology of Wound repair", editors Clark, RA.F. and Henson, P.M. Plenum Press, New York, p.p. 3-33.

Gabbiani, G., Hirshel, B J., Ryan, G.B., Statkov, P.R. and Majno, G. (1972). Granulation tissue as a contractile organ. A study of structure and function. J. Exp. Med. 135: 719-734. Horrigan, S., Campbell, J.H. and Campbell, G.R. (1988). Effect of endothelium on β-VLDL metabolism by cultured smooth muscle cells of differing phenotype. Atherosclerosis. 71: 57-69.

Kurkinen, M., Vaheri, A, Roberts, P.J. and Stenman, S. (1980). Sequential appearance of fibronectin and collagen in experimental granulation tissue. Lab. Invest. 43: 47-51.

Leibovich, S.J. and Ross, R. (1975). The role of the macrophage in wound repair: A study with hydrocortisone and anti-macrophage serum. Am. J. Pathol. 78: 71-100.

Roberts, AB., Sporn, M.B., Assovan, R.K., Smith, J.M., Roche, M.S., Heine, U.F., Liottay, L., Falanga, V., Kehrl, J.H. and Fancie, AS. (1986). Transforming growth factor beta: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collage formation. Proc. Natl. Acad. Sci. USA 83: 4167-4171.

Selye, H. (1953). On the mechanism through which hydrocortisone affects the resistance of tissues to injury. An experimental study with the granuloma pouch technique. J. Am. Med. Assoc. 152: 1207-1213.

Claims

CLAIMS: '

1. A topical composition comprising vitreous or an extract thereof fro mammalian ocular tissue wherein said vitreous or its extract preferentiall accelerates growth of granulation tissue compared to skin tissue.

2. A topical composition according to claim 1 further comprising one or mor pharmaceutically acceptable carriers and/or diluents.

3. A topical composition according to claim 1 or 2 further comprising one o more wound healing promoting agents and/or antimicrobial agents.

4. A topical composition according to claim 1, wherein the ocular tissue is fro a livestock animal.

5. A topical composition according to claim 4 wherein the livestock animal i selected from die list consisting of bovine, ovine, equine, porcine species and goat

6. A topical composition according to claim 5 wherein the livestock animal is bovine species.

7. A topical composition according to claim 1 immobilized or otherwis impregnated onto a solid support.

8. A topical composition according to claim 7 wherein the solid support is bandage, dressing, gauze or sutures.

9. A topical composition comprising vitreous or an extract thereof fro mammalian ocular tissue wherein said vitreous or its extract preferentially stimulate in vitro growth of vascular smooth muscle cells and/or myofibroblasts whil substantially not stimulating growth of skin fibroblasts and/or keratinocytes.

10. A topical composition according to claim 9 further comprising one or more pharmaceutically acceptable carriers and /or diluents.

11. A topical composition according to claim 9 or 10 further comprising one o more wound healing promoting agents and/or antimicrobial agents.

12. A topical composition according to claim 9 wherein the ocular tissue is from a livestock animal.

13. A topical composition according to claim 12 wherein the livestock animal is selected from the list consisting of bovine, ovine, equine, porcine species and goat.

14. A topical composition according to claim 13 wherein the livestock animal is a bovine species.

15. A topical composition according to claim 9 immobilized or otiierwise impregnated onto a solid support.

16. A topical composition according to claim 15 wherein the solid support is a bandage, dressing, gauze or sutures.

17. A topical composition according to claim 1 or 9 in lyophilized form and which is optionally reconstituted in aqueous medium prior to use.

18. A method for enhancing or accelerating healing of a wound in an animal, said method comprising contacting said wound witii an effective amount of the composition according to claim 1 or 9 for a time and under conditions sufficient to promote healing of the wound.

19. A method according to claim 18 wherein the animal to be treated is a human, livestock animal or companion animal.

20. A method according to claim 20 wherein the animal to be treated is a human.

21. A method according to claim 18 wherein the wound is healed by secondar intention.

22. A method according to claim 21 wherein the wound is an ulcer.

23. A method according to claim 18 wherein the wound is damage to dermal tissue.

24. A method according to claim 19 wherein the wound is a bone fracture.

25. A method according to claim 18 wherein the wound to be healed is a ski graft.

26. A method according to claim 18 wherein the wound is a post-operative ski lesion lacking epidermis or dermis tissue.

27. A method according to claim 18 further comprising the topical application o another wound healing promoting agent and/or an antimicrobial agent.

28. A method according to claim 18 wherein the ocular tissue is from a livestoc animal.

29. A method according to claim 28 wherein the livestock animal is selected fro the list consisting of bovine, ovine, equine, porcine species and goat.

30. A method according to claim 29 wherein the livestock animal is a bovin species.

31. A method according to claim 18 wherein the composition is topically applie on a bandage, dressing, gauze or sutures.