Protein C in burns as a marker of severity, recovery and prognosis

Dr Ruilong Zhao1,2, Dr Thomas Lang1, Dr Albert Kim2, Dr Rachel McGrath2, Dr Meilang Xue1, Dr Haiyan Lin1, Mrs Susan Smith3, Dr Siobhan Fitzpatrick2, Dr Nancy Huang2, Dr Andrew Zimmerman1, Dr Aruna Wijewardena2, Prof Gregory Fulcher2, Dr John Vandervord2, Prof Chris Jackson1

1Sutton Laboratory, Kolling Institute at Royal North Shore Hospital, St Leonards, Australia, 2Royal North Shore Hospital, St Leonards, Australia, 3Raymond Purves Laboratory, Kolling Institute at Royal North Shore Hospital, St Leonards, Australia


The natural course of some severe burns patients is difficult to predict without robust biomarkers to assist traditional evaluation. We have recently shown that plasma protein C (PC) levels are reduced following burn injuries (Lang et al. 2019). Here we elucidated PC’s potential utility as a dynamic marker of injury severity and extent, and value as an early predictor of clinical outcome.

We enrolled 86 patients in a single-centre prospective cohort study conducted at Royal North Shore Hospital in Sydney. Serial blood samples were taken and analysed for PC and a panel of six pro-inflammatory cytokines including CRP. Clinical outcome was determined by a previously described composite of fluid/ICU/surgical requirements termed “increased support”.

Plasma PC and CRP both tracked injury recovery over time. Notably, PC was markedly reduced on Day 0 before recovering to normal levels. Throughout the first 1-2 weeks, PC was consistently lower in patients with larger burns (p<0.01), deeper burns (p<0.01), and those with inhalational burns (p<0.05). Whereas in CRP these differences were not apparent until 3-6 days after injury. At baseline on Day 0, burn size (p<0.0005), depth (p<0.0005), inhalational injury (p<0.0005), and PC levels (p<0.0005) – but not any other inflammatory mediator tested – were predictors of clinical outcome. Multivariable regression showed Day 0 PC predicted increased support even when adjusted for burn size, depth and presence of inhalational injury (OR 0.879 (0.793-0.973), p=0.013), with 78.9% sensitivity and 92.3% specificity.

PC may therefore provide a simple yet powerful adjunct for assessment of the burns patient.


Lang, C, Zhao, R, Kim, A, Wijewardena, A, Vanderword, J, McGrath, R, Fitzpatrick, S, Fulcher, G & Jackson, C 2019, ‘Plasma protein C levels are directly associated with better outcomes in patients with severe burns’, Burns, (accepted 01/05/2019)


Ruilong is a junior doctor and a final year PhD student based at the Royal North Shore Hospital in Sydney. There he has discovered a keen interest in burns and reconstructive surgery. His research focuses on protein C’s actions in wound healing and as a biomarker in burn injuries.

Amniotic membrane as a scaffold: harvesting, preparation and the effects of preservation

Dr Susan Jehangir1, Dr Seng Chua2, Dr  John Harvey1, Dr  Peter Maitz3, Dr Zhe Li3

1Children’s Hospital At Westmead, Westmead, Australia, 2Department of Women’s and new born health Westmead Hospital, Westmead, Australia, 3Burns Unit, Concord Hospital, , Concord, Australia



There is a renewed interest in the use of Amniotic membrane (AM) as a scaffold for the ex vivo expansion and delivery of cells. This study investigates the issues with harvesting and effects of different preservation procedures on the properties of the AM to serve as a scaffold for the adherence and expansion of stem cells.


Fresh AM was harvested from seronegative term pregnant mothers undergoing elective Caesarean section deliveries under sterile conditions. Decellularization of human amnion HAM was carried out by incubating the tissue in hypotonic tris-EDTA buffer containing EDTA and aprotinin at 4 oC for 16 h. Then tissue was then transferred to a 0.03% SDS solution and incubated, with shaking, for 24 h at room temperature. After treatment with SDS, the tissue was washed thoroughly in buffered saline and either cryopreserved or lyophilised for storage. Cells were seeded onto the epithelial side with medium changed every 2 days. The effects of cryopreservation and lyophilisation were evaluated on mechanical and histological characteristics of the AM and its effects on cell adherence and expansion.


Preliminary results favour the freeze dried AM for cell adhesion and expansion. Final results awaited


Visiting from Christian Medical College, Vellore as a short term trainee. Fellow at the Children’s Hospital at Westmead with a special interest in the use of amniotic membrane in burns

Preclinical assessment of efficacy of Wharton’s jelly mesenchymal stem cells on a scaffold of amniotic membrane in wound healing using a porcine model.

Dr Susan Jehangir1, Dr  John Harvey1, Dr  Peter Maitz2, Dr Zhe Li2

1Children’s Hospital At Westmead, Westmead, Australia, 2Burns Unit, Concord Hospital, , Concord, Australia



This project studies the regeneration of epidermal and dermal components with the use human Wharton’s Jelly Mesenchymal Cells (WJ-MSCs) on a scaffold of Freeze Dried Human Amniotic Membrane (FD-HAM) and Cryopreserved Human Amniotic Membrane (CP-HAM) in porcine excisional wound model


Fresh AM was harvested from seronegative term pregnant mothers undergoing elective Caesarean section deliveries under sterile conditions. The membrane was de-cellularised  and either cryopreserved (CP-HAM) or freeze dried (FD-HAM) for storage. Commercially available WJ-MSCs  were seeded on the different forms of the decellularised membrane. Six, 5×5 cm cutaneous wounds using a dermatome at 0.8mm setting, 2cm apart will be created on the dorsum of 20-25kg Great White pigs on either side of the spine under general anaesthesia. The treatments (Wound control, WJ-MSCs alone, FD-HAM, CP-HAM, FD-HAM + WJ-MSCs, CP-HAM+ WJ-MSCs)   were allocated by Latin squares to randomise the application. A barrier dressing were applied. Wound healing was followed for 28 days using wound imaging and histology at euthanasia. The healing wound was assessed by Bates Jenson score and the healed wound by the Vancouver Scar scale. The final histology will assess the epithelization, sweat and hair follicle regeneration and fibrosis in the wounds.


The study is in progress. Results are awaited.


Susan is a fellow at the Children’s Hospital at Westmead  visiting as a short term trainee from Christian Medical College Vellore, India

Preclinical assessment of efficacy of Wharton’s jelly mesenchymal stem cells on a scaffold of Aloe vera-Polycaprolactone in wound healing using a rat burn model.

Dr Susan Jehangir1, Ms Sowmya Ramesh1, Dr  Madhuri Walter1

1Christian Medical College, Vellore, India


Background:  This project studies the regeneration of epidermal and dermal components with the use human Wharton’s Jelly Mesenchymal Cells (WJ-MSCs) on a Aloe vera-Polycaprolactone (A-PCL) composite scaffold and Collagen sheet  in a rat burn model

Methods: MSC from Wharton Jelly were isolated, cultured and characterized using flow cytometry. Cells at passage 1 (1×105 cm2) were seeded on an in-house fabricated aloe vera and collagen sheets (Kӧllagen®, India). Critical sized (4 cm2) in vivo burn model in rat was established by applying a heated coin over the back for 30 sec without external pressure. Experimental groups (n=7 each) were a. burn control, b. collagen alone, c. aloe vera alone, d. collagen +WJ-MSCs and e. aloe vera +WJ-MSCs. Outcome was assessed by gross appearance and histology (H&E).

Results: Culture expanded MSC were characterised positive for human cell surface markers. The wound remained unhealed in the burns control group at day 28 with a Bates Jensen score of 18. The other four arms healed with the fastest healing occurring in the A-PCL with WJ-MSCs group. Histology of the burns control group showed the non-healing ulcer with minimal epithelization and no sweat or sebaceous glands. The A-PCL group showed epithelization with no adnexal structures. Both collagen groups showed a similar picture with complete epithelization and a few hair follicles and additionally, immature sweat glands in the collagen WJ-MSC group. The A-PCL WJ-MSC group showed complete epithelization with abundant hair follicles and sweat glands.

Conclusion: We found that in-house fabricated aloe vera sheets with MSC augmented skin repair. There was significant regeneration of skin appendages in this group.


Susan is a consultant Pediatric surgeon at the Christian medical College at Vellore. She is currently a fellow at the Children’s Hospital at Westmead.

Exploring the Heterogeneity of Keloid Fibroblasts

Dr Andrew Stevenson1, Ms Nicole Hortin1, Dr. Philip Melton2, Professor Fiona Wood3, A/Prof Mark Fear1

1Burn Injury Research Unit, University Of Western Australia, Crawley, Australia, 2Centre for Genetic Origins of Health and Disease , Perth, Australia, 3State Burns Service, Fiona Stanley Hospital, Murdoch, Australia


Keloid scarring is a fibroproliferative disorder of the skin of unknown pathophysiology, characterised by fibrotic tissue that extends beyond the boundaries of the original wound. The main cell type that produces extracellular matrix (ECM) proteins during the healing process is the fibroblast, and a dysregulation of these cells during wound healing is what leads to the excessive collagen deposition in keloids. Fibroblasts have been poorly characterised until recently and were once thought to be relatively homogenous (Hu et al., 2018).

Fibroblast heterogeneity has recently been explored using single cell RNA-seq (scRNA-seq) which sequences RNA from individual cells, differentiating cells by their transcriptome. scRNA-seq has been used to identify sub-populations of cells in many tissues, including skin (Tirosh et al., 2016; Tabib et al., 2018; Philippeos et al., 2018). However, there has been little phenotypic characterisation and to date there has been no published data on keloid fibroblasts.

We have performed scRNA-seq to identify subpopulations of fibroblasts using keloid tissue isolated from elective surgery patients, with preliminary analysis identifying 5 distinct fibroblast populations, confirming that keloids are heterogeneous and suggesting that subpopulations within the keloid are driving scar progression. This work has implications for not only keloids but broader treatment of scars, demonstrating that not all scar cells are alike. Future work will involve phenotypic characterisation of cell subtypes in order to devise more precise, tailored treatment for keloid scars.


Hu MS, Moore AL and Longaker MT. (2018) A Fibroblast Is Not a Fibroblast Is Not a Fibroblast. Journal of Investigative Dermatology 138: 729-730.

Philippeos C, Telerman SB, Oulès B, et al. (2018) Spatial and Single-Cell Transcriptional Profiling Identifies Functionally Distinct Human Dermal Fibroblast Subpopulations. Journal of Investigative Dermatology 138: 811-825.

Tabib T, Morse C, Wang T, et al. (2018) SFRP2/DPP4 and FMO1/LSP1 Define Major Fibroblast Populations in Human Skin. Journal of Investigative Dermatology 138: 802-810.

Tirosh I, Izar B, Prakadan SM, et al. (2016) Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science 352: 189.


Andrew Stevenson is a postdoc working at the Burn Injury Research Unit at the University of Western Australia. He was awarded his PhD in 2016 titled “Investigating the role of epigenetics in scar maintenance” from UWA, and has been lucky to continue on this and other research focused on the mechanisms and treatment of fibrosis at the Burn Injury Research Unit.

Andrew was born and grew up in Perth, and attended the University of Western Australia at which he studied a BSc in Biochemistry and Molecular Biology, graduating with honours in 2007. After a gap year travelling Europe, he began working at the Burn Injury Research Unit as a research assistant, where he took up the offer of a PhD in 2011. Outside of science he enjoys playing guitar, wakeboarding and playing soccer.

The Impact of Matrix Stiffness on Fibroblast Phenotype and Keloid

Zhenjun Deng1, Manon Subilia2, Dr. Yu Suk Choi1, Nicole Hortin1, Dr. Andrew Stevenson1, FRACS Fiona Wood1,3, A/Prof. Cecilia Prêle1, Dr. Mark Fear1

1The University Of Western Australia, Perth, Australia, 2Swiss Federal Institute of Technology Lausanne, Lausanne, Switzerland, 3Fiona Stanley Hospital, Perth, Australia


Keloid is driven in part by a feedback loop in which the fibrotic extracellular matrix is both a cause and consequence of fibroblast matrix deposition. Yes-associated protein (YAP) is a key mechanoactivated coordinator of fibroblast phenotype and matrix production and is translocated to the nucleus upon activation. Here, we have studied the impact of matrix stiffness on fibroblast phenotype by culturing fibroblasts on a stiff coverslip (>109 Pa), 2 kPa polyacrylamide (PA) hydrogel (normal dermis-equivalent stiffness), 20 kPa PA hydrogel (keloid-equivalent stiffness), and 40 kPa PA hydrogel (extreme pathological-equivalent stiffness). In this study, we found that matrices of pathological stiffness induced YAP nuclear translocation as did stimulation with TGF-β in normal fibroblasts. However, in contrast, keloid fibroblasts showed high and stable YAP activation which does not respond to increasing matrix stiffness, suggesting that keloid fibroblasts have lost the normal mechanism of mechanosensation. In addition there is no increase in nuclear YAP in response to increasing stiffness nor to treatment with TGF-β, suggesting keloid fibroblasts are autoregulated to produce higher levels of activated YAP irrespective of biological and physical cues.

Taken together, this data shows that normal skin fibroblasts respond to the physical properties of the matrix in vitro. However, keloid fibroblasts appear to have elevated activation of mechanotransduction pathways irrespective of the micro-environment. This change may significantly contribute to the pathogenesis of keloid and has implications for potential treatments to ameliorate keloid disease.


My name is Zhenjun Deng. I am a second year PhD student from the University of Western Australia. My PhD is focused on cells and extracellular matrix interaction and how these may be important in keloid pathology.

Platelet reactivity increased following non-severe burn injury

Mr Blair Johnson1, Mr Andrew Stevenson1, Ms Fiona Wood1,2, Mr Mark Fear1, Mr Matthew Linden1

1University Of Western Australia, Crawley, Australia, 2Department of Health, Perth, Australia


It has been established that burn survivors are at a long-term increased risk of cardiovascular disease, with increased lengths of stay when hospitalised. However, the underlying pathology is poorly understood; this is compounded by a lack of research into non-severe burn injuries (NSBI), which comprise the majority of all burn cases in Australia (84%).

Platelets have an established role in the pathogenesis of cardiovascular diseases. During the acute burn injury circulating pro-thrombotic mediators increase susceptibility to venous thrombosis and pulmonary embolism. It has not been determined whether there is an association with NSBI and altered platelet reactivity that could drive a life-long risk of cardiovascular disease.

Platelet function was assessed in the post-acute period by flow cytometric analysis of platelet activation in mice and humans. Blood was collected from mouse models of NSBI (8% TBSA) and sham controls then incubated with a panel of canonical platelet agonists. Increased reactivity to stimulation of platelet collagen receptor was observed (1.2 fold change from sham). We then followed up in adult humans presenting with a NSBI, collecting blood samples at 2- and 6-weeks post-injury. An increase in circulating monocyte-platelet aggregates was observed without stimulation at 2 weeks, and expression of the fibrinogen-binding site upon stimulation with threshold doses of collagen-related peptide at 2 weeks. Platelet responsiveness to collagen-related peptide showed a trending increase across dosages and time points.

Platelet reactivity has been demonstrated to remain altered after the acute burn injury has resolved. Further research is recommended to establish a causative mechanism.


Blair has just begun his PhD at the University of Western Australia investigating changes to immune profile and platelet function after burn injury.

Dr Bronwyn Dearman


Bronwyn Dearman, a Medical Scientist at the Adult Burns Unit, Royal Adelaide Hospital has been instrumental in establishing the first Skin Laboratory for SA providing both a clinical service and the development of the Biodegradable Temporising Matrix (BTM) (a product now in fledgling global use in burn communities). To complement this first stage and assist in abolishing the need for a skin graft, the development of the Composite Cultured Skin substitute (CCS) encompasses her current research interests.

Associate Professor Mark Fear


A/Prof Mark Fear completed his PhD at the Royal London Hospital in dermatology, finishing in 2003. Since 2007 he has worked with Professor Fiona Wood on burn injury, scarring and fibrosis. His current research is focused on understanding the molecular and cellular pathogenesis of scarring and fibrotic disease, and  the development of novel therapies targeting fibrosis.

Dr Shiva Akbarzadeh


Dr Shiva Akbarzadeh is an expert in skin tissue engineering with a proven record in autologous skin culturing for transplantation. She was responsible for manufacturing CEA on a gel carrier to treat major burns in a clinical trial at the Alfred 2013-2016. She is experienced in experimental design and analysis and has established a novel methodology for skin tissue engineering with clinical relevance. She has also established a mouse model for wound healing using skin substitutes.



ANZBA is a not for profit organisation and the peak body for health professionals responsible for the care of the burn injured in Australia and New Zealand. ANZBA encourages higher standards of care through education, performance monitoring and research.

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