Measuring the harvesting of keratinocytes for wound healing

Contract research in support of the development of a medical device

BT&C Inc.

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A medical device company had the ambition to develop a wound healing product that involved transplanting patient skin to their own wound.  Autologous skin grafts were nothing new, but the approach was definitely novel and much less intrusive than traditional cut and paste grafting. 

Normal skin is a three layer matrix of cells and proteins.  The outer layer, or epidermis, consists of keratinocyte skin cells on the surface which is backed by a basal layer of protein and fibroblasts (connective tissue cells).  The next layer down is the dermis which contains blood and lymph vessels, hair follicles and sweat glands.  The third layer down, the subcutaneous, is composed of collagen and fat cells. 

Large wounds such as burns heal from the periphery inward.  When skin is damaged, it is the fibroblasts that rush to the rescue by populating the open wound.  These cells deposit collagen which further stimulates their growth.  The wound is consequently populated by a fibroblast and collagen matrix.  The resulting repair does not have the same properties as normal skin.  This is scar tissue and not only looks different, but functionally it is inferior to normal skin.  Grafting skin over open wounds is one way to minimize scars however this creates additional trauma if taken elsewhere from the patient.

Research has shown that wounds populated by both keratinocytes and fibroblasts create an environment which allows for proper cell growth and differentiation to re-create damaged skin.  Indeed living bandages have been developed that layer a living keratinocyte-collagen-fibroblast sandwich over wounds.  The bandage provides the environment that allows the patient to re-populate the wound correctly.  Unfortunately, such living bandages have been shown to be extremely cost prohibitive.  This cost factor was the client’s rationale for developing this new wound treatment.

In a nutshell, the new method involved plucking small pieces of epidermis from the patient, using a device like an ultra-fine cheese grater, and then dispersing the pieces on the wound like grass seed.  The donor site for the “seeds” healed readily (it is similar to a scrape) and initial testing by the client demonstrated this process worked on animal models.  With a successful testing of proof-of-concept, optimization of the process was pursued.

BT&C’s role in this project was to develop a methodology for measuring the effectiveness of different harvesting devices.  Like many projects undertaken by BT&C, there were no methods to reference.  Thus, working from prior knowledge and logic, it was hypothesized that a harvesting method which minimized tissue destruction and maximizes subsequent growth of keratinocytes in vitro would produce the best “seeds” for re-populating a wound.

To test this hypothesis first required baseline data on keratinocytes.  The animal healing models were performed on pigs, thus swine keratinocytes were isolated from skin sections and expanded in vitro.  These cells were used to study morphology and to enumerate cells by lysing cells and measuring the released lactate dehydrogenase.  Keratinocytes were also grown from skin explants in order to provide a visual guideline.

Freshly harvested skin “seeds” were assayed for cell death and growth potential.  Harvested “seeds” were tested for LDH activity which provided a measurement of cell death during collection.  The “seeds” were also plated into 24 well tissue culture plates with keratinocyte growth medium.  After an incubation period, keratinocyte growth was assessed microscopically and then enumerated by lysing the cells and measuring the released LDH.

The data collected by BT&C was referenced with “seed” performance in animal trials.  The combination of the work performed by BT&C and the animal modeling performed off-site was applied to selecting the best harvesting apparatus.