Obtaining Growth Factor Techniques

Genetic engineering pursues the modification of hereditary patterns by introducing or selecting one to several genes that correspond to an organism of a different species (these may be in bacterial, plant or animal eukaryotic cells) in order to synthesize a desired protein.

These advances have not only permitted the purification of the different growth factors but also the production of large quantities of these proteins through the following genetic engineering techniques:

a. Gel Electrolysis. It identifies proteins depending on their electric charge.

b. Markers. Recognizable segments of DNA along the gene chain which allows one to estimate the relative location of other genes.

c. ELISA Technique. (Enzyme Linked Inmuno SorbentAssay) It recognizes the spatial zone configurations of proteins or nucleic acids.

d. Gene Amplification. It is obtaining numerous copies of a specific DNA fragment and is done through the Southern Blot technique and the Polymerase Chain Reaction method (PCR).

e. Recombinant DNA. It consists of the precise reading of DNA nucleotide sequences and cutting with restriction enzymes in precise and known locations. Recombinant DNA sequences may be obtained through various methods: chemical synthesis, copying of messenger RNA, DNA polymerase, etc .

Plasma Rich in Autologous Factors (PRFC)

It is an autologous mixture, obtained by the patients’ blood centrifugal, which allows degranulation of platelets –with the growth factor liberation found in the cytoplasm-. Through blood centrifugal, one also obtains Plasma Poor in Platelets (PPP), which is the residual plasma that is obtained in the second part of this procedure, which contains coagulation factors, mainly the fibriogeneous one. Its function in the last stage of coagulation is that of transforming into fibrin, a previous activation through the trombine and calcium molecule. The fibrin mesh allows platelet catching and stabilization of blood coagulation, contributing also to speedy and effective scarring of soft tissue that cover the area to be regenerated.

Fig. 215-4 Technique for obtaining growth factors

Obtainment Procedure

One draws patient blood (between 10 and 40 ce), depending on the amount of Plasma Rich Platelets (PRP) and Plasma Poor in Platelets (PPP) required, knowing that for every 10 ce of blood, one obtains approximately 1 cm. of PRP and 1 cm. of PPP (Fig. 215- 4A). The blood is poured into sterile tubes that contain 3.8% sodium citrate used as an anticoagulant (Fig. 215-4B).

One places the mixture in the centrifuge for 5 minutes at approximately 350 degrees Celsius, and obtains a separation of fluid according to the density of its three basic components (from lower to higher density): the Plasma Poor in Platelets (PPP) is acellular plasma; Plasma Rich Platelets (PRP), the component with the highest concentration on platelets, therefore the highest concentration of FC. Later, comes a layer of white blood cells and finally red blood cells. (Fig. 215-4C, 215-4D y 215-5).

It has recently been clinically proven that a small portion of the superior layer of red blood cells contains less mature platelets that are also larger in size; therefore they are also included in the PRP (this is why it possesses a pink color).

Afterwards, one extracts plasma rich in growth factors by penetrating the area with highest concentration of platelets with a pipette (Fig. 215-4E).

Lastly, one injects rich plasma into the designated area of the growth factors while the patient is under general anesthesia or neuroleptoanalgesia .

Use in Various Medical Arenas

The potential for clinical use of growth factors are many. In hemato-oncology, hematopoyetic growth factors have been applied to eritropoyetin for example, a FDA drug approved for the treatment of different types of anemia: the stimulating factor of granulocyte colonies (G-CSF) and the stimulating factor of granulocyte and macrophage colonies (GM-CSF) used in myelosuppression and other cytopenias secondary to chemotherapy; the interpherons for treatment of malign entities. In oftalmology topical Epidermis Growth Factor (EGF) has been used in corneal and conjunctive damage. In otorrinolaringology, Epidermis Growth Factor (EGF) and the Fibroblast Growth Factor (FGF) have shown efficacy in remodeling the eardrum membrane.

Fig. 215-5. Blood components distributed by molecular weight.

In neurology, Nervous Growth Factors (NGF) have been used in cases of degenerative disorders such as Alzheimer and in traumatic nerve damage treatment In endocrinology the Insulin type Growth Factor (IFGI) is still in its experimental stages but being used to treat stunted growth due to hormonal issues as well as in cases of mellitus diabetes. In cardiovascular surgery intramuscular viral vectors have been injected with plasmids of the Vascular Endothelial Growth Factors (VEGF) in order to improve peripheral circulation. Satisfactory results have been published on the use of VEGF leading to reduction of pain (chest angina) and angiographic improvement verified via tomography technology. So far, there have been no concerns regarding the toxicity of topical growth factors in early experimental clinical trials (Phase I and II). However, it is necessary to tract these results over time since they have been studied only for the last 5 to 10 years. The FDA approval process for healing agents is slow, mainly due to strict regulations in order to satisfy requirement necessary for approval.

Use in Plastic Surgery, Factors of Growth Factors in Chronic Wounds

Among chronic open wounds that will not heal or have delays in the reepitelization process, there are lesions, neuropathies in the lower extremities of diabetics, wounds or lesions due to pressure, ulcers produced by venomous stasis, and wounds that do not heal properly in patients with compromised immune systems. Even though many theories have taken place to explain the causes that do not allow proper wound healing, it has not been possible to establish a singular criterion and the causes may very likely be multifactorial.

There are several circumstances associated to pathologies or habits that interfere with the biological sequence that allows the healing process to take place, these are:

Local factors such as infections, strange matters, ischemia, trauma, radiation, local toxins, artery and venous insufficiency, hyperthermial; other systemic actors such as nutritional deficiencies, diabetes, hepatic diseases; some habits such as alcoholism and smoking tobacco 13'14-15; use of medications such as Corticoids and chemotherapy agents, other circumstances include racial factors, wound location and method employed which may negatively affect healing processes in elective surgeries. Being able to recognize the causes of the healing malfunction will assist in avoiding future possible complications.

Published studies on Recombinant Growth Factors in chronic wounds show contradictory results, possibly due to the lack long term of follow up. Brown, et al, states that the topical treatment of skin donor locations with FCE accelerates the rate of epithelial regeneration in all patients on an average of 1.5 days, using PCE every 12 hours through a silver sulfadiazine transporter. Falanga, et al 17, published a double blind study on 44 patients with venous ulcers without obtaining significant differences between those patients who received the topical and those in the control group.

Chronic diabetic ulcers secondary to occlusive sickness of large vessels frequently require surgical interference. On the other hand, ulcers in lower extremities secondary to micro vascular alteration such as in the case of diabetes, the neuropathologic disease and/or venous stasis are not frequently docile when treated with revascularization. The repeated infection, eschemia, and trauma make contribute to the development of chronic ulcers.

All of these non healing lesions have shown extremely low levels of growth factors, its use has been studied in ulcers of diabetic patients of different etiologies, however, the best results have been observed in europatic ulcers 18.

The majority of these studies have used a healing agent derived from autologous platelets (autologous FCHDP), which mainly contains platelet forming factor -4, FCDP, FCT-J3 and FCE. In blind studies 3le the FCHDP used as a topical treatment accelerates the ce of healing 19. On the contrary, the derived platelet growth factor (DPGF) in an isolated manner, also applied topically, did not show significant improvements 20.

Jean Louis, et al21, states that a pilot study with Fibroblastic Growth Factor (FGF) in diabetic ulcers does not show significant differences compared to treatment with placebo, explaining therefore the lack of effects by the alterations that diabetics present in wound healing, one which probably requires the combination of various growth factors such as the synergetic effect with insulin 22.

The administration of various growth factors, including recombinant isomer DPGF and bFGF have demonstrated clinical improvement in animals and humans 23, in clinical studies in phase III and have been accepted by the FDA for their clinical use. In 3 out of 4 multicentric clinical studies, in order to evaluate the efficacy of Becaplermin®, a growth factor derived from human recombinant platelets (rhPD-GF-BB) showed an increase of healing speed in patients with neuropathic ulcers when compared to the control group. Becaplermin is in fact the first growth factor approved by the FDA to be used in the treatment of diabetic neuropathic ulcers 2324.

In terms of lesions from pressure, it has been observed that the prevention and alteration of the local strengths such as the local debridement when prescribed are of great help. However, recent clinical studies with the growth factor derived from human recombinant platelets (recombinant PDGF-BB) have demonstrated promising results in accelerating the curing and healing process through advanced pressure 25-26-27.

Patients who have received chronic treatment with corticosteroids, or those who have received chemotherapy or radiotherapy present great alterations of healing. In these patients, the application in animal tests of TGF-beta 1 reverts this situation 28.

On the other hand, patients with an active infection or those who have been in deep sepsis present significant delays in the healing process. In these septic patients, the neutralization of the proinflammatory cytokines and growth factors responsible for the healing delay may favor the treatment .

Growth Factors in Hypertrophic Scars and Keloids

In the skin lesions, the result of an excessive response o exaggerated process of normal scarring process is the formation of hipertrophic scars, keloids, and contracture.

By definition, hipertrophic scars are elevated but do not extend past the border of the wound, and they retract occasionally over time; keloids however, grow past the borders of the wound and may continue to grow because the healing process continues to be active. Both conditions are associated with an excessive inflammatory response and or prolonged one and to the production of extra cellular matrixes, including the increase of collagen deposits and decrease of its metabolism.

The cytokines and growth factors play an important roll in the pathology of keloides and hypertrophic scars 29.

The beta transformative growth factor (TGF-[beta])j seems to have the most intense effect in promoting the synthesis of collagen that favors fibrosis, and elevated levels of mRNA, of TGF-[beta] 1 in keloides and hypertrophic scars have been found 30 31. One has been able to establish that TGF-[beta], and TGF-[beta]2 promote scarring, while TGF-[beta]3 may reduce it. The antibody administration that neutralize the effect of TGF-[beta]j and TGF-[beta]2 as well as the exogenous administration of TGF-[beta]3 have been used in order to diminish the scarring activity in rats, which could be applied in order to diminish the activity of the hypertrophic scars or of the keloids 32 .