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¡¡Exploration of Fundamental Mechanisms of Adult Human Body Regeneration

1. Intrinsic regenerative potential of human body clinically validated by MEBO
2. Potentially Regenerative Cells (PRCs) in every tissue and organ of the body
3. The regeneration process recapitulates embryonic development process and mimics scar less wound regeneration of the fetus
4. Regenerative condition and environment
  4.1 Unique mode of debridement via liquefication of necrotic tissues to minimize injury to viable tissues
  4.2 Formation of a respiratory membrane on the wound
  4.3 A unique mode of controlling and preventing bacterial infection without using conventional antibacterial agents
  4.4 Creating and sustaining physiological moist environment to promote regeneration
  4.5 Clinical techniques for creating a regenerative local environment and condition

3. The regeneration process recapitulates embryonic development process and mimics scar less wound regeneration of the fetus

By using tissue samples obtained from different time points during the regeneration process of the wounds in burn patients, Dr. Xu and his team obtained precious evidence showing dynamic changes of cells at both the cellular and tissue levels.  By using immunohistochemical labeling of specific biomarkers, results were obtained that indicates that adult wound regeneration process under the conditions provided by using MEBT/MEBO recapitulates embryonic development and mimics scarless wound healing of the fetus.

Compared to fetal wound healing, the wound healing process in an adult with fully developed organs is carried out in a relatively more hostile environment than that for a fetus. The wound is susceptible to adverse effects caused by "normal" inflammatory response of the body to wounding and by exogenous agents such as bacteria that causes infection and further inflammation systemically and on the site. As discussed above, the prevailing thought in the art is that adult wound healing must be scarring healing because the adult-type, "normal" inflammatory wound healing is evolved to reduce the risk of infection at the expense of healing quality.  The compelling results obtained in the clinical practice using MEBT/MEBO change this dogma by showing that an fully developed adult possesses an inherent ability of self-repair and regeneration in response to wounding if suitable conditions are provided exogenously, and the wound healing process can mimic that occurring in a fetus at the early gestation stage to result in scarless healing in severely damaged skin.

Figure 6.3.1 shows the representative changes in the cells and tissues in a full-thickness burn wound of a patient (Figure 6.3.1a) treated with MEBT/MEBO. Figure 6.3.1b shows that 24 hours post burn injury there was coagulation and necrosis of epidermis and degradation of collagenous fibrous in superficial layer of the dermis. In the presence of the MEBO wound ointment, the necrotic tissues in the superficial layer is liquefied and discharged gradually. Because the necrotic tissues are liquefied instead of being surgically removed, the remaining viable tissues are protected from secondary injury caused by surgery.

On day 4 post burn there was already high activity of regeneration at the wound site with active proliferation of cells in the interstitial subcutaneous tissue (Figure 6.3.1c).  On day 7 post burn a large number of regenerative cells appeared at the wound site (Figure 6.3.1d).  On day 14 post burn massive regenerative cells appeared and nascent tissue started to form at the wound site (Figure 6.3.1e&f).  On day 21-28 post burn wound regeneration was near complete.  The wound was covered by stratified squamous epithelium; blood vessels, nerve and most of the appendages such as hair folicles started to form in the dermis (Figure 6.3.1g&h).  The skin was regenerated with normal structure as shown (Figure 6.3.1i).  At a higher magnification under an electron microscope, the junction between the dermis and epidermis is completely natural. Moreover, collagenous fibers in the new skin are arranged in a normal order three-dimensionally.  See Figure 1.2.2.9.

To monitor the dynamic changes of cells in growth and differentiation during the process of adult tissue and organ regeneration in vivo, wound regeneration of full-thickness burns is used as a clinical model to demonstrate how adult cells respond to exogenous agents under the treatment using MEBT/MEBO.

Dr. Xu and his research team demonstrated for the first time that embryonic epidermal stem cells are induced or activated and able to proliferate in adult human under conditions favorable for physiological tissue repair and organ regeneration. Such regenerative conditions are provided by using MEBT and by applying MEBO wound ointment topically to the wounds.

During the treatment of the patients with full-thickness burns, vigorous cellular activity on the wounds was observed in the presence of MEBO wound ointment.  These ¡°hyper-active¡± cells (Figure 6.3.1.c&d) are confirmed to be cells expressing keratin-19 detected by immunofluorescent staining with a monoclonal antibody against human keratin-19 (K-19).
 
 Figure 6.3.2 shows typical dynamic changes in the level of K-19 expressing regenerative cells monitored at different time points during wound regeneration of a patient under the treatment of MEBT/MEBO. 

The results summarized in Figure 6.3.2 show that in the normal adult epidermis, few cells were K-19 positive (Figure 6.3.2a). In contrast, for skin in the wounds, there was a moderate amount of regenerative epidermal stem cells that were stained positive for K-19 24 hr post burn (Figure 6.3.2b). On day 4 post burn (Figure 6.3.2c), the number of K-19 expressing cells increased around the sweat gland, capillaries and hair follicles.  On days 7 and 14 epidermal stem cells continued to increase, reaching peak values during this period (Figure 6.3.2d and 6.3.2e, respectively). Until days 21 and 28, the number of K-19 expressing cells decreased to low levels (Figure 6.3.2f and 6.3.2g, respectively).

On day 30 post burn, electron microscopic examination of the sections taken from the new skin of the patient revealed that the skin regenerated by using the methodology of the present invention retains its normal, physiological structure. Also, the collagenous fibers in the regenerated new skin were normal in both size and spatial arrangement, measured 0.1-05 m and with characteristic light and dark periodic cross striation (64 nm). See Figure 1.2.2.9.


Figure 6.3.2. Stained frozen tissue sections using mouse anti-human keratin type 19 (K-19) monoclonal antibody (x200 magnification). a: Normal skin (No K-19 positive cells. b-g: After treatment with MEBO wound ointment. b: 24 h postburn (a few K-19 positive cells appeared). c: Day 4 postburn (the number of K-19 positive cells increased). d: Day 7 postburn (the number of K-19 positive cells peaked). e: Day 14 postburn (the number of K-19 positive cells plateaued). f: Day 21 postburn (the number of K-19 positive cells decreased). g: Day 28 postburn (the number of K-19 positive cells decreased significantly).  See Xu (2004) in Burns Regenerative Medicine & Therapy, p.117, S. Karger AG, Basel, Switzerland.

Where do K-19 expressing cells come from?

In burn wounds of deep 2nd degree or higher, epidermal stem cells residing in the basal layer of epidermis are destroyed. More interestingly and challengingly, in burn wounds of full-thickness burn, the whole epidermis and dermis are destroyed with only the hypodermis, the fatty layer of the skin, remaining viable.  Treatment of full-thickness burn wounds with conventional methods such as skin grafts or skin substitutes results in wound-closure with disfiguring scars and substantial loss of normal function of appendages of the skin and physiological networks of blood vessels and nerve.

However, as shown above, adult patients who sustained full-thickness burns, after treated with MEBT/MEBO, could recover with skin regenerated with substantial restoration of its structures and function. What is the source(s) of epidermal cells that compose to form the tissues that constitute the regenerated skin organ?

The results obtained in the treatment of burn patients reveal that at least part, if not all, of the epidermal cells are originated from embryonic epidermal stem cells expressing K-19.

Keratin-19 has been known to be a biochemical marker of skin stem cells emerged during the embryonic development.  In normal adult human epidermis, there is no or few K-19-expressing keratinocytes present.  Michel et al. (1996) ¡°Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: Keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage¡± J. Cell Science 109:1017-1028.  Hsia et al. (2008) ¡°Effects of topically applied acitretin in reconstructed human epidermis and the Rhino mouse¡± J. Investigative Dermatology 128:125-130.

As shown in Figure 6.3.2, the number of cells stained positive for K-19 increased dramatically while the body underwent active tissue repair and skin regeneration, and the number declined when the regeneration was complete. It is believed that these embryonic epidermal stem cells proliferated and differentiated to produce specific types of cells, including other types of keratinocytes expressing keratin 1, 9, 10, and 16 that moved upward towards the epidermis.  The detection of high expression of the embryonic epidermal stem cell marker K-19 indicates that the regeneration of skin by using MEBT/MEBO may recapitulate embryonic development and mimic the scarless wound healing of the fetus.

Histological analysis of tissue samples taken from many patients reveals that the wounds treated with MEBT/MEBO manifest distinct characteristics of cellular proliferation, differentiation and migration that resemble those of early-gestation fetal wound healing.  In the presence of MEBO, activation or induction of regenerative stem cells, including K-19 expressing epidermal stem cells, is followed by proliferation and directional differentiation of the stem cells into specific types of cells necessary for the regeneration of a fully functional skin including blood vessels, hair follicles, collagenous fiber, interstitium and nerves. 

These experimental findings are groundbreaking and will have a profound impact on our understanding of the regenerative capacity of the human body.  To our best knowledge, this is the first time embryonic epidermal stem cells are detected during the wound regeneration process in an adult human body.  These stem cells are originated from adult somatic cells that are induced to behave like embryonic epidermal stem cells under the conditions provided by MEBO wound ointment and by using MEBT.  Such induction occurs in situ and in vivo, i.e., at the wound site where the cells are cultured in a moist regenerative environment provided by the ointment.  Neither stem cell transplantation nor genetic engineering is involved in this process.

In comparison, for a long time in the field of regenerative medicine a great deal of efforts has been focused on studying individual factors (wound healing factors and genes) contributing to regeneration. For example, many in the field are trying to grow and control proliferation and differentiation of embryonic stem cells ex vivo, hoping to replace defective/diseased cells/tissues with cell therapy (Murry & Keller (2008) ¡°Differentiation of embryonic stem cells to clinically relevant populations: Lession from embryonic development¡± Cell 132:661-680), or to genetically manipulate adult somatic human cells to mimic embryonic stem cells, such as those induced pluripotent stem (iPS) cells (see review by Nishikawa (2008) ¡°The promise of human induced pluripotent stem cells for research and therapy¡± Nat. Rev. Mol. Cell Biol. 9:725-729; and commentary by Cyranoski (2008) ¡°5 Things to know before jumping on the iPs bandwagon¡± Nature 452:406-407).

Although human somatic cells can be rendered by exogeneous gene transfer to look more like stem cells, so far iPS cells differentiate chaotically to form teratomas in vitro.  Although the adenovirus-transduced with transcription factors could convert mouse exocrine cells into insulin-producing beta cells, no functional pancreatic islet has formed by these genetically programmed cells. Zhao & Daley (2008) ¡°From fibroblasts to iPS cells: induced pluripotency by defined factors¡± J. Cell Biochem. 105:949-955.

While these studies of embryonic stem cells and iPS cells enrich our knowledge about the biological functions and pliability of the human cells, currently practical applications of these cells to regeneration of the human body are still at various early stages of research and development. 

By contrast, MEBO has demonstrated that by using a completely different approach¡ªbotanical-based products with superior proven safety profiles¡ªto achieve regeneration of tissue and organ in situ and in vivo through induction of normal somatic cells residing at the site of tissue/organ damage to behave like stem cells.  As shown above, the result is natural, physiological proliferation and differentiation of tissue following the body¡¯s intrinsic genetic lineage, providing just enough multiple cell types to replace the lost or injured cells so as to restore its structural and functional integrity. Compared to the approach of ex vivo cultivation of human embryonic stem cells or iPS cells whose clinical efficacy and safety are yet to be determined, MEBO¡¯s innovative approach with proven clinical success represents a more direct and safer way to help the body regenerate when needed.

 
   
¡¡ACHIEVEMENTS
Introduction
Core Science & Technology of MEBO
Exploration of Fundamental Mechanisms of Adult Human Body Regeneration
Eradication of Cancer Cells via Regeneration
 









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