Collagen membranes for skin wound repair: A systematic review

Abstract

Membranes or skin dressing are common treatments for skin wound injuries, collagen being one the most effective materials for their manufacturing. Many different sources of collagen with diverse methods of extraction and processing have been used, with evidence of positive effects on the stimulation of skin wound healing. In spite of these factors, there is still limited understanding of the interaction between collagen membranes and biological tissues, especially due to the series of different types of collagen origin. In this context, this study aimed to conduct a systematic review of the available literature examining the effect of various collagen membranes for accelerating skin wound healing in experimental animal models and clinical trials. The present review was performed from March to May of 2020 searching in two databases (PubMed and Scopus). The following Medical Subject Headings (MeSH) descriptors were used: “collagen”, “dressing”, “membranes”, “skin” and “wound”. After the eligibility assessment, 16 studies were included and analyzed. The studies demonstrated that collagen was obtained predominantly from bovine and porcine sources, by acetic acid and/or enzyme dissolution. Additionally, most of the studies demonstrated that the membranes were processed mainly by freeze-drying or lyophilization methods. All the in vivo and clinical trial studies evidenced positive outcomes in the wound healing process, thus confirming that collagen membranes are one of the most efficient treatment for skin wounds, highlighting the enormous potential of this biomaterial to be used for skin tissue engineering purposes.

Keywords

Collagen wound dressing membranes skin

Introduction

Skin wounds are very common injuries, caused by many different reasons, including physicochemical or thermal damage and metabolic diseases such as diabetes. They result in functional imbalance, increased level of pain, disability or even death.¹,² It is estimated that approximately 5.7 million people in US are affected by wound lesions, with the treatment cost exceeding USD 50 billion dollars annually.³

Skin wounds can be divided into acute and chronic wounds. Acute wounds, in general, heal by themselves in a smooth and efficient way.⁴ Conversely, in the presence of some specific situations such as bad nutrition, associated diseases, age or extensive wound extension, the process of skin regeneration may be impaired, resulting in chronic or non-healed wounds.⁵,⁶

Many treatments for chronic skin injuries have been proposed such as surgery, pharmacological interventions, use of growth factors and stem cells, and wound dressing with or without membranes.⁶,⁷

Acute wounds normally heal in an orderly and efﬁ-

cient manner. They progress smoothly through the

four distinct, but overlapping phases of wound healing:

hemostasis, inﬂammation, proliferation and remod-

elling

Acute wounds normally heal in an orderly and efﬁ-

cient manner. They progress smoothly through the

four distinct, but overlapping phases of wound healing:

hemostasis, inﬂammation, proliferation and remod-

elliAcute wounds normally heal in an orderly and efﬁ-

cient manner. They progress smoothly through the

four distinct, but overlapping phases of wound healing:

hemostasis, inﬂammation, proliferation and remod-

elling

Membranes are one of the most efficient treatments for skin injuries, serving as protection from infection, reducing the level of pain, and stimulating and supporting cell migration and proliferation.⁸,⁹ It is important to emphasize that many different biomaterials serve as raw material to be used for membrane manufacturing such as auto-grafts,¹⁰ synthetic materials (artificial polymers), and natural materials like chitosan and collagen.⁸,¹¹

Collagen (formed by a triple-stranded helix of proline and glycine polypeptide chains) is the most abundant protein in vertebrates and is involved in the structural integrity of many tissues such as ligaments, tendons and bones.^12–15 As a biomaterial, collagen is biocompatible, bioactive and has low immunogenicity, making it extremely suitable for tissue engineering and regenerative medicine, or TERM, strategies for human health issues, including membranes for skin wounds.^16–19

In this context, it has been demonstrated that collagen membranes are resorbable (with an absorption rate matching tissue ingrowth), bioactive, have porosity and are able to support cell growth.¹⁷,²⁰ For this reason, they have been frequently used for treating skin wounds.^18–20 Many different sources of collagen for membrane manufacturing have been explored, including bovine and porcine bone and skin, marine species such as fish skin, marine sponges and jellyfishes.²¹ Collagen membranes, manufactured based on different methods, have demonstrated positive outcomes for stimulating tissue healing.^16–18,²² Table 1 shows some examples of collagen membranes commercially available used for skin wound healing.

Table 1.

Collagen membranes/wound dressing commercially available.

Company/product	Collagen type	Setting characteristics and applications^a	Biosafety and biocompatibility according to FDA^b
FIBRACOL® – Systagenix	Bovine collagen type I	Skin dressing for pressure injury; venous and arterial vasculogenic ulcers; diabetic ulcers and second-degree burns.	FIBRACOL® has been demonstrated to be an acceptable topical wound dressing according to the following safety testing: Agar Overlay Assay (L929 cells); Hemolysis (Rabbit RBCs); MEM Elution Test; Muscle Implantation (Rabbits); Systemic Injection (Mice); Rabbit Pyrogen Assay; Primary Skin Irritation (Rabbits).
SURGIMEND® – INTEGRA LIFESCIENCES	Bovine collagen type I and III	Applied for General, Plastic and Reconstructive, and Trauma surgeons.	The device was characterized in a pre-clinical, acute/subacute animal model to evaluate the effect of meshing on the host biologic response after implantation.
MILTEX® – INTEGRA LIFESCIENCES	Bovine collagen type I	For application to moist or bleeding clean oral wounds created during dental surgery, to control bleeding and to protect the surface of the wound from further injury.	The membrane is intended for fixation or stabilization of guided tissue regeneration membranes to bone at the surgical site.
HELISTAT® – INTEGRA LIFESCIENCES	Porcine collagen	Absorbable collagen hemostatic sponge for wound dressing.	Helistat is indicated in surgical procedures (other than ophthalmological and urological surgery) as an adjunct to hemostasis when control of bleeding by ligature or conventional procedures is ineffective or impractical.
INTEGRA® – INTEGRA LIFESCIENCES	Bovine collagen type I	Bi-layer structure composed by silicone sheet, bovine collagen and chondroitin sulphate. Indicated for dermal regeneration.	Integra Dermal Regeneration Matrix is indicated for use in the treatment of partial and full-thickness neuropathic diabetic foot ulcers that are greater than 6 weeks in duration, with no capsule, tendon or bone exposed.
PRIMATRIX® – INTEGRA LIFESCIENCES	Fetal bovine dermis	Acellular dermal repair scaffold.	PriMatrix is intended for the management of wounds that include partial and full thickness wounds; Pressure, diabetic, and venous ulcers; Second-degree burns; Surgical wounds – donor sites/grafts, post-Moh’s surgery, post-laser surgery, podiatric; wound dehiscence; Trauma wounds – abrasions, lacerations and skin tears; Tunnelled/undermined wounds; Draining wounds.

^aAccording to the manufacturer's information.

^bAccording to the U.S. Food and Drug Administration.

Although the evidence points towards positive effects of collagen membranes on skin wound healing, the wide range of collagen sources and the lack of standard protocols for membrane manufacturing (with many different extraction methods, chemical fixation agents, etc.) make it difficult to directly compare the published results. Also, the mechanisms by which collagen membranes interact with skin wounds are still controversial, and for many, their use as a treatment modality remains highly-debated.¹² Therefore, it is important to closely analyze the available data from the literature in order to improve the understanding of the effects of this therapeutic intervention on skin healing under different experimental conditions, to determine its safety and efficacy.

In this context, the purpose of this study was to perform a systematic review of the literature of studies investigating the effects of collagen membranes (from different origins) on improving skin wound healing in in vivo experimental models and clinical trials.

Methodology

Review protocol

This systematic review was performed according to the Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) guidelines.²³ The search was conducted from March to May 2020 using the PubMed and Scopus databases. The search was carried out according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. The following descriptors of the Medical Subject Headings (MeSH) were used: “collagen”, “dressing”, “membranes”, “skin” and “wound”. In addition, several synonyms in the titles and abstracts were searched for each component.

Study selection

Two reviewers (ACMR and TAA) analyzed independently the title and the abstract of the selected works and identified the potential studies using the inclusion and exclusion criteria. The two reviewers had access to the selected studies to verify eligibility. Disaccords were solved by discussion. The selected studies were further reviewed during the full-text screening. Those that did not follow the eligibility criteria were excluded.

Inclusion criteria

In vivo experiments with animal models and clinical trials with patients with skin injuries or burns treated with collagen membranes.

Type of intervention: use of collagen membrane as treatment for skin wound healing.

In addition, articles were included by active search in bibliographic reference list of the selected articles and search in personal archives.

Articles must be written in the English language and published in the last 30 years.

Exclusion criteria

Studies of characterization of the membrane, in vitro studies, in situ studies, reviews, and case reports.

Studies without skin wounds, burns or injuries.

Lack of description of the skin wounds, burns or injuries, methodology or outcomes.

Animal models or clinical trials with systemic diseases (such as diabetic rats).

Data extraction

The variable analyzed to determine the quality of evidence was based on the histological analysis of the in vivo studies. In addition, the species/strain, animal sex, age, weight, origin of membrane, skin wound type, wound size, implantation period, ways of treatment, collagen extraction protocol, membrane manufacturing technique/crosslinking method, physical and morphological characteristics, collagen origin, analysis performed in the in vivo studies, outcome measures, analysis performed in the clinical trials, results and histological outcomes were also included in our analysis.

Types of reported results

Due to the heterogeneity of the primary studies, it was not possible to perform a meta-analysis. In order to compare the effect size (ES) of both techniques, we calculated the normalized average difference considering the values before and after the intervention. They were further classified as small (<0.20), moderate (about 0.50), or large (>0.80), according to Cohen criteria.

Quality of the evidence was determined using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach, which analyzes the following domains: trial design limitations due to risk of bias, inconsistency of results, indirectness, imprecision of results, and publication bias.

Results

The flow diagram demonstrates the search strategy used in the present study (Figure 1). A total of 49 articles was retrieved from the databases (31 from PubMed and 18 from Scopus). Duplicated records were excluded (n = 21). The remaining 28 full-text articles were assessed for eligibility and from those eight were excluded. Additionally, four studies were excluded for other reasons. There were 16 studies finally included and analyzed in this systematic review.

Figure 1.

PRISMA flow diagram of search strategy.

Table 2 shows the characteristics of all included studies, such as author, species/strain, animal sex, age, weight, origin of membrane, skin wound size, implantation period (days) and way of treatment. Among the articles analyzed, seven studies used rats as the experimental model, such as Wistar rats^24–27 and Sprague-Drawley rats.^28–30 Two studies used rabbits,³¹,³² one study used dogs and cats,²² one used guinea pigs,³³ one used mices²⁰ and one minipigs.³⁴ The clinical trials included evaluated the effects of the membrane on the process of skin wound healing.²⁹,^35–37

Table 2.

Characteristics of the studies.

Author	Species/strain	Animal sex	Mean age	Weight	Origin of membrane	Skin wound type	Wound size	Implantation period	Method of treatment
Rana et al.²⁴	Wistar rats	Female	^a	100–150g	Collagen-chitosan blend (shrimp shell chitosan + rabbit skin collagen)	Incision burns	2.5 cm diameter	16 days	Membranes were placed at the region of the injury with 1 ml of collagen hydrogel applied on a daily basis.
Balaure et al.²⁵	Wistar rats	Male	8 weeks	350 ± 10 g	Bioactive dressings based on collagen and zinc oxide	Third degree burns	1.5 cm²	3 weeks	Membrane was applied to the burn.The evolution of the skin burns was monitored daily.
Li et al.²⁶	Rats	Male	8–10 weeks	150 g	Col, N-Col, Col-GO and N-Col-GO hybrid membranes	Incision wounds	20 mm diameter incision	14 days	Membranes were placed at the region of the injury and changed on alternate days.
Shi et al.³²	New Zealand White rabbits	^a	^a	2.5 kg	Porous sponge scaffolds (one prepared by collagen derived from porcine skin and another prepared from fish scales)	Scald area	2 x 2 cm	28 days	Membranes were placed at the region of the injury and changed once every 3 days.
Kaasi et al.²²	Dogs and cats	Male and female	>8 years	^a	Collagen type I biomembrane	Skin lesions of different etiologies considered difficult to heal	> 2 cm	120–240 days	Every 2 days, the gauze tape was removed and the wound was cleaned with physiological saline. Extra alginate hydrogel was applied, as needed, and covered up again with fresh gauze tape to prevent infection.
Xie et al.²⁷	Rats	Male	^a	200–250 g	Chitosan-collagen-alginate composite dressing	Full-thickness incision wounds	0.8 cm	15 days	After covering with the membranes, the wounded rats were soaked in seawater at a constant temperature of 28 °C for 4 h. Rats were monitored twice daily.
Byun et al.³⁶	Humans	Men and women	64.4 ± 2 years	^a	Rapiderm® -porcine collagen membrane	Two standardized full-thickness skin wounds	^a	3–4 weeks	The donor site was covered with the vascularized deep fascia to prevent the exposure of flexor tendons. The first dressing change, excluding the removal of the tie-over dressing, was performed 5 days postoperatively. The grafted site was treated with a closure dressing and maintained by an extension volar splint for 2–3 weeks.
Petersen et al.³⁴	Göttingen minipigs	Female	39 weeks±12 days	22.6 ± 1.4 kg	Matriderm®- collagen-based matrices	Full-thickness skin defects	2.0 cm diameter	21 days	Wounds were covered with a single application of the new collagen–gelatin
Wang et al.²⁸	Sprague-Dawley rats	Male	2 months	225 ± 16 g	Polyurethane membrane/knitted mesh-reinforced col–chitosan	Two standardized full-thickness skin wounds	2 cm	12 weeks	Membranes were placed at the region of the injury. Two weeks after, the upper layers of the dermal substitutes were removed, and other full thickness skin was excised. Then the split-thickness skin was used to cover the proximal wounds.
Amal et al.³³	Guinea pigs	Male	^a	1 kg	Chitosan-collagen membrane	Excision wound	2 cm	25 days	Membrane of aqueous extract of Punica granatum was implanted in the wound
Aoki et al.²⁰	Mice	Male	^a	25–30 g	VitriBand® with collagen sponge	Full-thickness round wounds	15 mm diameter	14 days	Membranes were placed at the region of the injury and replaced every day to prevent contamination. The membrane was replaced every other day for wound exudate control.
Potocká et al.³⁷	Humans with no severe accompanying diseases	Both genders	18–65 years	^a	Coladerm H/HM - collagen and hyaluronic acid based biosynthetic dermal skin	Patients requiring split-thickness skin grafting	^a	1st assesment 21 days, then 6 months with cosmetic treatment	Coladerm membranes were placed at the region of the injury. After 3 and 6 months, membranes were removed, and Coladerm H/HM with hyaluronic acid were placed with cosmetic treatment and scar formation evaluated.
Chen et al.³⁰	Sprague-Dawley rats	Male	^a	300–350 g	Chitosan membrane containing collagen I nanospheres	Full-thickness retangular wounds	2 x 2 cm	7 and 14 days	Membranes were placed at the region of the injury.
Bao et al.³¹	Rabbits	^a	^a	^a	Agar/collagen membrane	Cut skin	Diameter over 1.5 cm	3 weeks	Membranes were placed at the region of the injury.
Horch and Stark³⁵	Humans	Both genders	Similar age between the groups	^a	TissueFascie® and OpSite®	Burn, trauma, ulcer and tumor	^a	12.5 ± 3.4 days	Membranes were placed at the region of the injury and covered with a Vaseline gauze.
Gao et al.²⁹	Sprague-Dawley rats/Humans	Female rats/Humans both genders	Rat age not provided/Humans 3–38 years	Rats 250–350 g/Human weight not provided	Porcine dermal collagen and petroleum jelly dressing	Scald burn in rats/Burn, post burn and plastic surgery in humans	Rats 4 x 4 cm/Humans 50 cm²	Rats 10 days/Humans 16 days	In rats the wounds were covered with porcine dermal collagen membrane immediately after injury.In humans the area within the split skin thickness donor site was covered with a collagen membrane, 50 cm², and the remaining area was covered with petroleum jelly gauze.

^aInformation not provided in the study.

Table 2 also shows that half of the studies analyzed in this review applied the membrane directly to the wound, without dressing changes.²⁷,^29–31,^33–35 Seven studies performed more than two changes of membranes during experimental period ²⁰,²²,²⁴,²⁶,²⁸,³²,³⁶,³⁷ and one also included the use of cosmetics in association with the membrane.³⁷ Different wound sizes were used ranging from 15 mm² to 50 cm².²⁹ In addition, different timepoints were analyzed with a minimum experimental period of 7 days³⁰ and a maximum experimental period of 240 days.²²

A summary of collagen extraction protocols, membrane manufacturing processes as well as some of the physical and morphological properties of collagen membranes are listed in Table 3. We found that in nine studies, commercially available collagen was used (one from tendons of rats, four from bovine tissues, one from porcine tissue and for the others, the origin was not provided),²²,^25–27,³¹,^34–37 while five studies extracted collagen from bovine bone,²⁶,²⁸,³⁴,³⁵,³⁷ four studies used porcine-collagen,²⁰,²⁹,³²,³⁶ and two studies used collagen from marine fish.³²,³³ Interestingly, Kaasi et al. used collagen from rat tail tendon²² and Rana et al. used collagen extracted from rabbit skin.²⁴ Four studies did not provide data on the origin of the collagen.²⁵,²⁷,³⁰,³¹ The protocols for collagen extraction varied; three studies used an acetic acid and pepsin enzymatic method,²⁴,²⁸,³² two studies employed an acetic acid method,²⁰,³³ one used a tris-HCl buffer with acetic acid-pepsin extraction methodology,²⁹ and one utilized collagen produced from a high voltage electrostatic field system.³⁰

Table 3.

Collagen extraction and membrane protocols.

Authors	Collagen extraction protocol	Membrane manufacturing technique/crosslinking method	Membrane manufacturing	Physical and morphological characteristics
Rana et al.²⁴	Rabbit skins were suspended in acetic acid (acetic acid soluble collagen) and the non-dissolved residue was re-suspended in acetic acid/pepsin solution (pepsin soluble collagen). The solutions were filtered, precipitated with NaCl, centrifuged and re-suspended in acetic acid. Finally dialyzed and freeze-dried.	Solvent casting method/no crosslinking agent was applied.	Chitosan/collagen blend membranes were prepared using the solvent evaporation method. Both chitosan and collagen were dissolved in acetic acid and neutralized with NaOH. Solutions were mixed and the resultant polymer blend was washed, and freeze-dried.	The highest value of water absorption (25.85%) and equilibrium water content (72.1%) were observed with the membrane composed of the equal concentrations of chitosan and Col.
Balaure et al.²⁵	Collagen was purchased commercially.	Solvent casting method/glutaraldehyde solution as a crosslinking agent.	Col/Orange oil with zinc oxide membrane was manufactured by mixing all the components and homogenizing them. After, glutaraldehyde (0.1%) was added as a crosslinking agent and the gels were placed into petri dishes and lyophilized.	Scanning electron microscopy (SEM) micrographs reveals that the pore sizes of the membranes ranged around 20 nm.
Li et al.²⁶	Bovine collagen type I (commercially available).	Freeze-drying method/EDC/NHS as a crosslinking agent.	N-Col-GO membrane, composed of graphene oxide (GO), bovine collagen type I (collagen) and antioxidant N-acetyl cysteine (NAC) was manufactured with the addition of acetic acid into GO and Col. Solution was homogenized, laid over a cylindrical mold, freeze-dried and crosslinked by EDC/NHS. 0.1 mg/mL NAC was added into the membrane. collagen membrane (collagen), NAC loaded collagen membrane (N-collagen) and Col-GO hybrid membrane (Col-GO) were made using the above method.	SEM analysis showed that membranes with Col, N-Col, Col-GO, and N-Col-GO had interconnecting pores covering most of the space in the membranes. The water retention ability of N-Col-GO (3750.00 ± 521.800%) and Col-GO membranes (4569.20 ± 920.289%) was significantly higher than that of the collagen membrane (2468.05 ± 680.375%) and N-collagen membrane (2965.68 ± 522.043%) due to the incorporation of GO.
Shi et al.³²	Porcine skin was inserted into a 0.25% trypsin solution and then dried, smashed, and stored at room temperature. Fish scale-derived collagen (FSC), was obtained by dissolving into an acetic acid solution, filtration and centrifugation. Acetic acid/pepsin was added, filtrated and centrifuged. Both solutions were mixed, centrifuged, acetic acid was added. The mixture was dialyzed and freeze-dried.	Freeze-drying method/glutaraldehyde solution as a crosslinking agent.	For the membrane manufacturing (PSC), the porcine skin collagen extracted was inserted into a HCl solution and stirred. The liquid was quickly poured into a mold and freeze-dried to obtain the membrane. The dried membrane was crosslinked in glutaraldehyde 0.25% solution, washed with ultrapure water and freeze-dried. The FSC membrane were prepared in the same way as PSC.	SEM images of the PSC and FSC membranes exhibited large pores with a diameter about 100–200 μm (both in the surface and in the cross-section). The PSC membrane showed laminar structures on the cross sections, which were not found in the FSC membranes.
Kaasi et al.²²	Rat tail tendon collagen type I (commercially available).	^a	The membrane was purchased commercially.	Membrane dimension was 8 × 3 cm (length and width), with a thickness ranging from 200 to 300 μm, and a regular pattern of depressions/elevations.
Xie et al.²⁷	Collagen was purchased commercially.	Freeze-drying method/no crosslinking agent was applied.	The chitosan-col-alginate (CCA) membrane was prepared by coating and vacuum freeze-drying method. Chitosan was dissolved in acetic acid and collagen was added. Solution was mixed, freeze–dried and inserted in a mold with alginate.	SEM image showed that the surface of the CCA was smooth and fibrous. The swelling ratios were 10 times of its dry weight, the porosity 65.91 ± 0.033% and the degradation index was 3.12 ± 0.01%.
Byun et al.³⁶	Porcine skin purified atelocollagen (99.83%) (commercially available).	^a	The membrane was purchased commercially.	Two sizes of the Rapiderm® membrane were used in the experiments (10 × 10 cm and 8 × 6 cm), with a thickness of 1 mm.
Petersen et al.³⁴	Bovine collagen type I (commercially available).	^a	Col-gelatin membrane was manufactured via a novel, standardized spinning procedure. The Matriderm® (commercially available) consists of a three-dimensional lyophilized collagen matrix coated with elastin hydrolysate.	The collagen-gelatin membrane consists of fibers with a bimodal distribution of about 2 and 10 mm diameter and a pore size of 35–70 mm.
Wang et al.²⁸	Type I collagen from bovine tendons extracted with trypsin digestion and acetic acid dissolution.	Freeze-drying method/EDC/NHS as a crosslinking agent.	Collagen and chitosan were dissolved in acetic acid solution. The collagen-chitosan solution was poured into a flat plate mold, containing poly(L-lactide-co-glycolide) meshes. The blend was left to stand, and was lyophilized, cross-linked and lyophilized again. At the end, a bilayer membrane was obtained.	SEM analysis showed that membranes had a typical bi-layered structure, with a tight junction between both layers and a stable porous microstructure.
Amal et al.³³	Fish skin was treated with NaOH solution, stirred and washed. After, butyl alcohol was added, the solution was washed again and acetic acid was added. Solution was filtrated, centrifuged and the supernatant collected. NaCl was added and centrifuged again. Acetic acid was added and the collagen solution was sonicated and lyophilized.	Solvent casting method/no crosslinking agent was applied.	Col-chitosan membrane was manufactured by adding 0.5 g of collagen to 0.75 g of chitosan into a solution of acid acetic. The mixture was filtrated, put in a plastic petri plate and kept in an oven at 60 °C overnight for drying and to form the membrane. The pH was neutralized with NaOH, the membrane was washed with deionized water and dried overnight at 60 °C.	SEM showed a high number of pores of the membrane. Tensile strength of 45.36 ± 0.64 MPa.
Aoki et al.²⁰	The porcine derived atelocollagen solution 0.5% was prepared by uniformly mixing equal volumes of porcine skin raw material with acidic solution 1.0% and a serum-free culture medium.	Gelation, vitrification, and rehydration/no crosslinking agent was applied.	For the collagen membrane manufacturing, 1.8 mL of atelocollagen solution was mixed with phosphate-buffered saline into a separable container comprising a cylinder and a plain bottom plate. The membrane was converted to a collagen xerogel membrane by revitrification on a separable sheet and sterilized with ethylene oxide gas.	The membrane depicted excellent transparency, mechanical strength and permeability to proteins with high molecular weights.
Potocká et al.³⁷	Bovine atelocollagen type I (commercially available).	^a	^a	Coladerm H/HM is a term for a hybrid membrane with a specific bubble macrostructure prepared.
Chen et al.³⁰	Collagen type I nanosized was produced using a high voltage electrostatic field system.	Freeze-drying method/genipin as a crosslinking agent.	The chitosan-collagen membrane was manufactured by adding a solution of heated chitosan to genipin solution, poured in a glass dish and quenched with liquid nitrogen. The samples were freeze-dried and neutralized by NaOH. Then, nanosized collagen particles were seeded into the porous membrane by the static seeding method.	SEM images reveal that the chitosan-collagen membrane structure was not homogeneous. The porosity was in the range of 46–58%.
Bao et al.³¹	Collagen type I (commercially available).	Freeze-drying method/glutaraldehyde solution as a crosslinking agent.	Agar/collagen membrane was obtained by dissolving agar solution in hot water and cooled to 60 ^◦C. Bovine type I collagen was added and the solution was left to dried in a mold, at 50 ^◦C.	SEM images showed the interconnected pores of the membrane, with a laminated structure. The agar/collagen membrane concentration ratio (4:3) showed a relatively higher stretch modulus, about 540–819 MPa.
Horch and Stark³⁵	Bovine collagen type I (commercially available).	^a	TissueFascie® membranes were purchased commercially	^a
Gao et al.²⁹	Collagen from porcine skin was extracted by the addition into a tris-HCl buffer solution (pH 7.5) containing NaCl. Acetic acid was added. The tissue was digested with pepsin in acetic acid and centrifuged. Soluble collagen was precipitated with NaCl, dissolved in acetic acid and stored.	Salt preparation/no crosslinking agent was applied.	Collagen membrane was prepared in a petri dish, by adding NaCl powder over the soluble collagen (salt precipitation). collagen membrane was washed with tris-HCl buffer, pH 7.5, overnight.	^a

^aInformation not provided in the study.

Three studies used commercially available collagen membrane in their experiments,²²,³⁵,³⁶ another three used chitosan/collagen blends,²⁴,²⁸,³³ one a chitosan/collagen/starch blend,²⁷ one a chitosan-collagen-alginate blend and one chitosan-genipin enriched with collagen nanoparticles.³⁰

For manufacturing of the membranes, most of the authors dissolved the raw materials in acid acetic, followed by freeze-drying or lyophilization.^24–28,³²,³³ Three studies used glutaraldehyde solution as a crosslinking agent in the manufacturing process.²⁵,³¹,³² Some authors used some specific protocols such as described: Aoki et al. employed a gelation, vitrification and rehydration methodology,²⁰ Gao et al. used a salt precipitation method,²⁹ and Petersen et al. developed a novel spinning procedure.³⁴ Five studies that used commercially available membranes did not present information about the manufacturing process.²²,^34–37

A wide range of physical and morphological membrane characteristics can be observed. Scanning electron microscopy was used in seven studies to show the porosity and surface structure of the membranes.²⁵,²⁷,²⁸,^30–33 One evaluated the water absorption and equilibrium water content of the membranes,²⁴ one analyzed membrane thickness (ranging from 200 to 300 μm),²² one evaluated water retention ability,²⁶ and one swelling ratios (10 times of its dry weight).²⁷ Six studies did not show detailed information about the membranes (commercially available products).²²,²⁹,^34–37

Table 4 demonstrates the results and outcomes of the in vivo experiments. All 13 studies performed a macroscopic evaluation through images from the area of the wound after treatment with collagen membranes.²⁰,²²,^24–28,³⁰,^32–34 For all studies, treated animals had a decreased wound area after the experiment and one reported subsequent reduced scarring.³³ Histological analysis demonstrated a faster reepithelization process,²⁰,²²,²⁵,²⁷,²⁹,³²,³⁴ reduced inflammatory reaction,³⁰ reduced scar formation^20,25,30 and a faster rate of tissue healing.^{26,28,30–32} Four studies performed immunohistochemistry analysis and results are as described: Li et al.²⁶ and Wang et al.²⁸ observed an increase of cluster of differentiation 31 (CD31) immunostaining. Xie et al.²⁷ observed enhanced immunostaining of epidermal growth factor (EGF), CD 31, and transformation and growth factor (TGF) while Aoki et al.²⁰ observed a decrease of connective tissue growth factor (CTGF) and anti-a-smooth muscle actin (α-SMA) immunostaining after 7 days, and a decrease of cluster of differentiation 45 (CD45), macrophage F4/80 receptor (F4/80) and proliferating cell nuclear antigen (PCNA) immunostaining after 14 days. Wang et al.²⁸ also performed the immunofluorescence analysis and observed an increase of CD31 expression. Additionally, they performed a mechanical test and observed a gradual increase of tensile strength after 12 days.

Table 4.

Results and outcomes of the in vivo experiments.

Authors	Collagen origin	In vivo analysis	Results	Outcomes
Rana et al.²⁴	Rabbit skin	Macroscopic evaluation	Collagen membrane treated animals produced better wound contraction (>70%), reepithelialization period with no scar formation after 16 days post-surgery.	+
Balaure et al.²⁵	Collagen was purchased commercially	Macroscopic evaluationHistological analysis	Collagen-zinc oxide treated animals demonstrated a wound healing rate of 94.75 ± 0.33% after 21 days and complete reepithelialization after 21 days.	+
Li et al.²⁶	Collagen Type I	Macroscopic evaluationHistological analysisImmunohistochemistry	Membrane treated animals presented a decrease of 77.0 ± 17.0% of the wound area 14 days post-surgery. N-acetyl cysteine-loaded graphene oxide-collagen (N-Col-GO) enhanced granulation tissue formation and significantly increased expression of cluster of differentiation 31 (CD 31) after 14 days.	+
Shi et al.³²	Porcine skin (PSC) and fish scales (FSC)	Macroscopic evaluationHistological analysis	PSC and FSC treated animals presented a complete process of healing after 28 days. PSC treated animals presented a higher reepithelialization process after 21 days.	+
Kaasi et al.²²	Collagen was purchased commercially	Macroscopic evaluationHistology analysis	Membrane treated animals presented with complete wound healing after 30 days, with an increase in the vascularization and new skin formation covered by fur after 240 days.	+
Xie et al.²⁷	Collagen was purchased commercially	Macroscopic evaluationHistological analysisImmunohistochemistry	Chitosan-collagen-alginate (CCA) treated animals demonstrated a decrease in the area of the wound of 90% after 13 days. CCA resembled normal skin after 13 days, promoted reepithelialization and well-organized granulation tissue, and repaired the epidermis and dermis. CCA was able to increase the expression of epidermal growth factor, CD 31 and transformation growth factor.	+
Petersen et al.³⁴	Collagen was purchased commercially	Macroscopic observationHistology analysis	Collagen-gelatin 150 g/m² (multiple applications) treated animals presented a completely closed wound after 10 days. Collagen-gelatin 150 g/m² (multiple applications) scaffold demonstrated a higher density of epidermal cells when compared to untreated groups.	+
Wang et al.²⁸	Bovine tendons	Macroscopic observationHistology analysisImmunohistochemistryImmunofluorescenceMechanical tests	PLGAm/Chitosan collagen solution (CCS) treated wounds had a residual wound area of 2.65 ± 0.16 cm and showed newly formed tissue through the entire scaffold after 14 days. PLGAm/CCS group was able to increase the expression of CD31. PLGAm/CCS tensile strength gradually increased reaching 0.78 ± 0.03 MPa after 12 weeks.	+
Amal et al.³³	Rachycentron canadum	Percentage of reepithelialization	Chitosan-collagen-starch membrane (CCSM) treated groups had reepithelialization percentage over 85%.
Aoki et al.²⁰	Porcine skin	Macroscopic observationHistology analysisImmunohistochemistry	Collagen treated groups showed complete wound healing after 14 days. VitriBand (VB) was able to promote a flat and well-differentiated epidermal layer formation. VB had a lower expression of connective tissue growth factor (CTGF) and anti-a-smooth muscle actin (α-SMA) after 7 days. VB had a lower expression of cluster of differentiation 45 (CD45), macrophage F4/80 receptor (F4/80) and proliferating cell nuclear antigen (PCNA) after 14 days.	+
Chen et al.³⁰	Collagen was purchased commercially	Macroscopic observationHistology analysis	CGC promoted a complete heal after 14 days. Chitosan membrane with collagen I nanospheres (CGC) effectively absorbed all exuded fluids from the wound after 7 days. CGC exhibited a slight inflammatory reaction, fibroblast formation and collagen synthesis after 7 days.	+
Bao et al.³¹	Collagen was purchased commercially	Macroscopic examinationHistology analysis	No signs of infection, liquid exudation or visible scar in the lesion site. Repair tissue was normal and little debris was left without degradation after 3 weeks.	+
Gao et al.²⁹	Porcine skin	Wound healing rateHistology analysis	Collagen treatment promoted a reepithelialization rate of 69.1% after 10 days. Collagen membrane was able to promote epidermal regeneration.	+

Table 5 shows the results and outcomes of the clinical trials included in the present study. The type of wound most reported were burn wounds²⁹,³⁶,³⁷ and only one study included ulcers, tumors and traumas.³⁵ The methodology applied in those studies included a split thickness skin from a donor site to use with the membrane to heal the wounds.²⁹,^35–37

Table 5.

Results and outcomes of the clinical trials.

Author	Membrane origin	Clinical trials	Analysis	Results	Histological outcomes
Byun et al.³⁶	Rapiderm® – porcine collagen membrane	Ten patients who had undergone single-stage radial forearm free flap reconstruction of soft tissue defect in the oral and maxillofacial area.	Degree of wrist range of motion, any sensory disturbance and scar dimension.	The use of the porcine collagen membrane in the donor site of the radial forearm free flap is an alternative approach with less morbidity. The defects of the forearm free flap donor site had completely recovered within 6 weeks without tendon exposure, loss of collagen membrane, and infection. There was no significant difference between preoperative wrist motion and postoperative outcomes, which were evaluated by four types of functional parameters.	+
Potocká et al.³⁷	Coladerm H/HM – collagen and hyaluronic acid based biosynthetic dermal skin	20 patients requiring split-thickness skin grafting to treat burn wounds.	Rate of epithelization, presence of secretion, color of donor site, cosmetic performance, scar formation and pain scale.	Coladerm H/HM showed positive results covering the split-thickness skin graft donor in 10 days compared to Dermazin® (Lek) + Acidum Aceticum 1%. The average of epithelized surface using Coladerm H /HM was 74% while using the Dermazin® (Lek) + Acidum Aceticum 1% (magistraliter) was 64%.	+
Horch and Stark³⁵	TissueFascie® (bovine collagen I membrane) and OpSite® (traditional polyurethane dressing)	The indications for split thickness skin grafting in the two groups (OpSite® vs. TissueFascie®) were: burns (3 vs. 3), trauma (5 vs. 3), ulcer (3 vs. 3), tumor (1 vs. 1).	Mean healing time of complete epithelialization of split skin donor and pain scale by visual analogue scale.	The median time from operation to the observation of complete healing was 7.5 ± 2.5 days for the donor sites dressed with the collagen membrane and 12.5 ± 3.4 days for the donor areas dressed with a polyeurethane film. The discomfort experienced by the two groups of patients was significantly less after wound coverage with collagen.	+
Gao et al.²⁹	Porcine dermal collagen and petroleum jelly dressing	Twelve burn patients were included in the clinical trial of porcine dermal collagen on skin donor sites.	Healing rate of the donor site.	Dressing with collagen membrane significantly enhanced wound repair with a complete reepithelialization by 10.3 days after injury.	+

Three of the studies used commercially available membranes.^35–37 Rapiderm® is a collagen membrane from porcine source,³⁶ Coladerm® is a membrane fabricated with collagen and hyaluronic acid,³⁷ and TissueFascie® is a bovine collagen I membrane.³⁵ Only one study fabricated a novel biomembrane manufactured from porcine dermal collagen and petroleum jelly dressing.²⁹

It is important to emphasize that all clinical trials showed positive results in the process of skin wound healing after covering the injury with collagen membranes, with a reduction in the time of healing in all studies compared to when a traditional membrane was used in the control group. Moreover, all the authors observed a complete healing process with the membrane treatment.²⁹,^35–37

Table 6 shows the quality of evidence for the use of collagen membranes for skin wound repair according to the GRADE approach. The table shows the degree of evidence of the studies evaluating the effects of the collagen membranes on skin wounds in the animal studies. After the analysis, it was observed that there is moderate evidence (considering the variable “histological analysis”) showing the efficiency of collagen membranes on skin wound healing.

Table 6.

Summary of findings: Collagen membranes sources versus control/sham.

Collagen membranes for skin wound repair
Outcome	Limitations	Inconsistency	Indirectness	Imprecision	Publication Bias	Trials	Intervention (n)	Comparison (n)	Significant difference or Statistics	GRADE level of evidence
Intervention: Collagen membranes × control/sham
Histological analysis						Balaure et al.²⁵	10	10	Yes
						Li et al.²⁶	8	8	Yes
						Shi et al.³²	1^a	1^a	Yes
						Xie et al.²⁷	8	8	Yes
						Petersen et al.³⁴	6^b	6^b	Yes
						Wang et al.²⁸	12	12	Yes
						Aoki et al.²⁰	5	5	Yes
						Chen et al.³⁰	6	6	Yes
						Bao et al.³¹	5 ^a	5 ^a	Yes
						Gao et al.²⁹	10	13	Yes
	Serious	No	No	No	Undetected					Moderate^c

^aRabbits.

^bMinipigs.

^cLarge_Effect: Large (+1).

Discussion

The present study evaluated the effects of different kinds of collagen membranes (used alone or in combination with other materials) on the process of healing in skin wounds in animal models and clinical trials. The results related to the biological data demonstrated that collagen membranes were able to accelerate skin wound healing, decreasing the time of wound resolution in both in vivo studies and clinical trials. Our systematic review also found that for the in vivo experiments, many different skin wound sizes and types (ranging from 15 mm² to 50 cm²) as well as different timepoints of evaluation (ranging from 7 to 240 days) were used.²⁰,²⁸ Acetic acid and pepsin enzymatic methods were used for collagen extraction. For membrane manufacturing, dissolution of the materials in acetic acid, followed by freeze-drying or lyophilization methods were applied. Different sources of collagen (porcine, bovine and fish) were used for membrane manufacturing. In addition, both commercially available collagen and lab-made membranes were employed.

The most commonly used raw materials to extract collagen were bovine²⁶,²⁸,³⁴,³⁵,³⁷ and porcine tissues.²⁰,²⁹,³²,³⁶ These sources produce type I collagen, which is the main protein of the mammalian extracellular matrix, thereby providing structural stability, strength and constituting an appropriate material for manufacturing skin membranes.³⁸ However, its usage is sometimes limited due to religious constraints related to avoidance of animal products and for the prevention of transmission of diseases such as bovine spongiform encephalopathy.¹¹ In this context, alternatives have been explored such as collagen from marine origin (e.g. fishes, marine sponges, jellyfishes).¹¹,²¹ Some authors have recently discovered that fish skin represents an excellent source for obtaining collagen and has been used as an alternative for mammalian collagen in many different applications such as cosmetics, tissue engineering and pharmaceutical uses.³⁹ We noted that collagen was frequently blended with other materials such as chitosan,²⁴,^26–28,³⁰,³³ graphene oxide,¹⁶ alginate,²⁷,³⁰ polyurethane,²⁸,³⁵ zinc oxide,²⁵ and hyaluronic acid.³⁷ This is a common strategy used to optimize some characteristics of the membranes such as mechanical strength and bioactivity.²⁵

We observed that the studies included in this review used different protocols for collagen extraction and most of them involved dissolution in acetic acid and/or enzymes (such as pepsin and papain).²⁰,²⁴,²⁸,²⁹,³²,³³ The literature describes a wide variety of collagen extraction processes from different sources, but emphasizes the importance of optimizing extraction conditions to achieve a higher collagen yield.³⁹,⁴⁰ For example, to obtain collagen from marine sponges, protocols involve the use of EDTA,⁴⁰ trypsin digestion and water acidified with CO₂.⁴¹ In addition, trypsin and acetic acid have been widely used for collagen extraction from skin, bones and tendons of rats, bovine and porcine as they are able to digest proteins without causing significant damage to collagen fibers, thereby guaranteeing high extractability rate.¹⁷,^42–44 However, it is important to stress that there is a continuous need to develop optimized protocols for collagen extraction, focusing on obtaining a faster, less expensive and more efficient process.¹⁷ In addition, special attention needs to be paid to the use of “green” chemical products, as there is high demand to reduce the use of hazardous substances for the extraction of natural materials.⁴⁵

The manufacturing process of the membranes used in this review mainly involved dissolving the materials in acetic acid, followed by freeze-drying or lyophilization. Freeze-drying have been used by many authors to create matrices, membranes and scaffolds with structures containing interconnected pores that resulted from nucleation and crystallization of ice, which are excluded and distributed into reticulations.^46–48 Some authors used crosslinking agents in the manufacturing process.²⁵,³² It is known that collagen based biomaterials are susceptible to mechanical failure during handling which limits their applicability as biomaterials.⁴⁴ Consequently, one of the strategies for improving the thermal stability and tensile strength of collagen samples is the inclusion of crosslinking agents.⁴⁹

Most of the authors demonstrated, by SEM, that the membranes present porosity. Some authors used commercially available collagen and membranes and did not present morphological data.²²,²⁹,^34–37

Additionally, according to both the in vivo studies and clinical trials, it was demonstrated that there were positive outcomes on the process of wound healing after the treatment with collagen membranes. Data from the in vivo experiments demonstrated that all of the collagen membrane treated animals presented faster wound skin healing, with a smaller injury area, higher deposition of granulation tissue, and stimulation of reepithelialization and neoangiogenesis. These findings are in agreement with theories saying that collagen membranes applied to the site of the wound serve as protection against infections and attractants of fibroblast cells.⁵⁰,⁵¹ Bohn et al. stated that one of the mechanisms of action of collagen membranes on the process of tissue healing is that collagen is capable of reducing the levels of metalloproteinases, acting as a sacrificial substrate for excessive proteases in a chronic wound.⁵² Also, Aoki et al. demonstrated that a collagen membrane promoted epithelization and modulated the inflammatory process in an experimental model of burns in rats.²⁰ The same positive outcomes were observed in the clinical trials, where there was faster healing in wounds treated with the membranes. It is also worthwhile pointing out that different skin wound models were studied by the different authors but the majority looked at burns or skin incisions. This highlights the effectiveness and flexibility of the collagen membranes to be used in skin wounds with different morphologies.

The outcomes of this review confirmed our initial hypothesis that collagen membranes (from diverse origins or blended with another materials) are able to accelerate the process of skin healing in different types of wounds. Consequently, this therapeutic intervention constitutes a promising treatment for burns and skin incisions, including their use in the clinical setting. However, some limitations of the present review needed to be highlighted. For example, in view of the differences in skin metabolism in healthy compared to compromised conditions (e.g. diabetes mellitus), the biological performance of collagen membranes might be different. This aspect has not been analyzed in this review. Additionally, some membranes present issues such as the need of dressing changes, which may cause discomfort or pain.

In conclusion, this review demonstrates that the different types of collagen membranes (manufactured only with collagen or in association with another material) were biocompatible and interacted well with skin tissue. The membranes offer protection to the wound and stimulate tissue growth both in in vivo studies and clinical trials, representing an efficient treatment for skin wounds. There is huge potential for this biomaterial to be used for skin tissue engineering purposes. Further research is required to evaluate the biological performance of collagen membranes in compromised situations.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Julia Parisi

Abdias Fernando Simon

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