Doxycycline Loaded Collagen-Chitosan Composite Scaffold for the Accelerated Healing of Diabetic Wounds

The current research work is doxycycline loaded collagen-chitosan composite scaffolds for the accelerated healing of diabetic wounds. Diabetes is a chronic metabolic disease characterized by elevated levels of blood glucose. The longer the time individual have diabetes, the higher the risk of complications, such as cardiovascular disease, nephropathy, neuropathy, retinopathy, and foot damage, et cetera.

Nerve damage in the feet and also poor blood flow to the feet increase the risk of various foot complications. If left untreated these wounds may ultimately require toe, foot, or leg amputations. Roughly, 25%patients with diabetes mellitus have the opportunity to build up a diabetic wound in their lifetime.

The therapeutic management of diabetic wounds remains a hurdle to date which needs to be eventually crossed. Even though the pathophysiology of diabetic wound is multifactorial, it is believed that local treatment with anti-inflammatory drugs could decrease the elevated levels of MMP-9 and reestablishes the cutaneous homeostasis framework arrangement and prompts healing in diabetic wounds. Doxycycline, the MMP-9 inhibitor, was chosen in this study to suppress these elevated levels of MMP-9, a major inflammatory molecule responsible for the continued inflammation of diabetic wounds.

On the other hand, biomaterials have fascinated humans for several decades and have been at the core of many game-changing medical breakthroughs for efficient drug and macromolecular delivery and development of tailor-made biomedical devices. Hence, with this brief introduction, we have prepared a doxycycline loaded collagen-chitosan scaffold to attenuate, to accelerate the delayed healing in diabetic wounds. This is Bharat Kumar Reddy, research scholar from Department of Pharmaceutics, JSS College of Pharmacy, Ooty, India.

Today we are here to look into the procedure, how to prepare doxycycline loaded collagen-chitosan scaffold for the accelerated healing of diabetic wounds. Let's have a glimpse into the procedure, how to prepare. We are going to prepare 1.2%of collagen solution, for which we are taking 1.2 grams of Type-I collagen, which is from rat tail origin.

To this, we are adding 100 ml of Millipore water and kept it aside for half an hour in order to allow the collagen to get bulge in water. In the next step, 0.8%of chitosan solution was prepared, for which we are taking 0.8 grams of medium molecular weight chitosan, and this was mixed with 100 ml of 1%acetic acid. This solution also, we have kept aside for half an hour.

After half an hour, both the solutions were subjected for mechanical stirring overnight at an RPM of 2000. To the overnight stirred collagen solution we are slowly adding one gram of doxycycline. And the solution containing this collagen and doxycycline was subjected for half an hour of stirring at an RPM of 2000.

To this solution we are slowly adding overnight stirred chitosan solution. And this, make sure it's labeled as physical mixture which contains doxycycline, collagen, chitosan. This physical mixture we have subjected for another half an hour of stirring at an RPM of 2000.

After half an hour these physical mixture were transferred into to respective aluminum molds, which is having a dimensions of two into two centimeter and a thickness of 0.5 centimeter. Later, these molds were transferred into a metal container. These metal container was containing the aluminum molds, was subjected for deep freezing.

After 24 hours, we can clearly see the solidified physical mixture containing doxycycline, collagen, and chitosan. These solidified physical mixture was further subjected for lyophilization. The lyophilization was performed at minus 85 degrees plus or minus four degree Celsius for 72 hours.

After 72 hours, the lyophilized material were taken out from the lyophilizer. And here we can clearly see the completely dried physical mixture, which is nothing but known as doxycycline loaded collagen-chitosan scaffold. These scaffolds were subjected for crosslinking In order to perform crosslinking, initially, MES buffer was prepared by taking 0.488 grams of MES and 15 ml of water, from which we are taking out 20 ml of MES buffer and transferring separately into your beaker.

To these 20 ml of beaker, 50 mg of doxycycline loaded collagen-chitosan was added. And the soaking of this scaffold was performed for half an hour. After half an hour, the doxycycline loaded collagen-chitosan scaffold was taken out from the 20 ml MES buffer.

Further, it was soaked in a solution containing 19.5 ml of MES buffer, 0.1264 grams of EDC, and 0.014 grams of NHS. The soaking was performed for four hours time interval for the completion of crosslinking. After crosslinking, the crosslinked doxycycline loaded collagen-chitosan scaffold was taken out from the solution and transferred it into a container, which was detailed in the procedure before, and it was dried separately.

Here we can clearly see the non-crosslinked and crosslinked scaffold, along with the same images. From the images it is clear that crosslinking of the scaffold resulted in decreased pore size due to the formation of strong intramolecular bonds. Male Wistar rat was taken and the two into two centimeter mark was drawn on the dorsal thoracic region.

Excision wound was created around the mark and wound tracing was taken on a OHP sheet. The trace of the wound was measured with the help of graphical method. The wound created on the animal was around 176 mm.

Later, the created wound was treated with crosslinked doxycycline loaded collagen-chitosan scaffold with the primary dressing and monitored for 21 days. Once the animal was recovered from the anesthesia, each animal were caged individually. And these animals were are fed with normal diet and water.

All the animals, after creating the wound, were divided into three groups in which each group contained six animals. Here we can clearly see the difference between zero day and seven day in which doxycycline loaded crosslinked scaffold wound reduction was around 48%And in this image we can clearly see the difference between the day 14 and day 21 of all the groups in which doxycycline loaded collagen crosslinked scaffold was healed around 99%The healed area was isolated and processed for histopathologic studies with the help of HMB staining. Here we can clearly see the reepithelized epidermis, on day 21, in the doxycycline loaded collagen chitosan scaffold.

Whereas in the control and crosslinked scaffold, nothing but placebo, still the epidermis had to be formed. Like our own study, the prepared doxycycline crosslinked scaffolds has exhibited many ideal properties, such as good mechanical strength, biocompatibility, sustained release water absorption, along with antibacterial and anti-inflammatory properties. These scaffolds can be an ideal dosis form for the treatment of diabetic wounds.