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\begin{document}

\title{Exploring the Potential of Bovine Colostrum as a Bioactive Agent in
Human Tissue Regeneration: A Comprehensive Analysis of Mechanisms of
Action and Challenges to be Overcome}



\author[1]{Pedro Rassier dos Santos}%
\author[1]{Rosana Basso Kraus}%
\author[1]{Patrícia da Silva Nascente}%
\affil[1]{Universidade Federal de Pelotas}%


\vspace{-1em}



  
  \date{June 16, 2024}


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\selectlanguage{english}
\begin{abstract}
Bovine colostrum is a rich source of bioactive compounds, including
growth factors, that may promote tissue regeneration. Studies suggest it
has potential therapeutic benefits in humans, but further research is
needed to fully understand its mechanisms of action. This study aims to
review scientific articles on the use of bovine colostrum as a bioactive
agent for tissue regeneration in humans and twelve articles were
selected. In vitro studies show that bovine colostrum can benefit cells
by reducing cell proliferation and inducing differentiation. The
concentration of colostrum used has a direct impact on the results, with
no positive results observed at some high concentrations (20\%).
Extracted portions of colostrum (EVs and IMF) showed better results than
the complete colostrum. In vivo studies show that bovine colostrum is a
promising natural source for wound healing, as its bioactive factors
help in the orderly progression of the healing process. However, some
studies did not find any benefit related to tissue regeneration, and
more research is needed to explore the full potential of colostrum in
regenerative medicine and to develop more effective therapies for wound
healing.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Wound healing involves the replacement of damaged tissue with newly
formed tissue and occurs in four phases: hemostasis, inflammation,
proliferation, and remodeling. The transition from the inflammatory to
the proliferative phase is a crucial step in wound healing, typically
occurring around day 4 (KIM et al., 2022; LAND\selectlanguage{ngerman}ÉN, LI \& STAHLE, 2016;
ZOMER \& ANDREA, 2018). This process is regulated by various cell types,
enzymes, cytokines, and growth factors (DONG \& GUO, 2021; KAUR; JOSHI;
SINGH, 2022).

Bioactive agents used in tissue regeneration include growth factors like
epidermal growth factor (EGF), transforming growth factor-beta (TGF-\selectlanguage{greek}β\selectlanguage{english}),
and platelet-derived growth factor (PDGF), which stimulate cell
proliferation, angiogenesis, extracellular matrix synthesis, and cell
differentiation. Additionally, substances such as antioxidants,
polyphenols, and omega-3 fatty acids have been studied for their effects
on tissue regeneration, including reducing oxidative stress and
modulating inflammation. The combined use of these bioactive agents can
increase the efficacy of tissue regeneration and promote faster and more
complete recovery (TANIDEH, et al., 2017; JONES et al., 2019)

Bovine colostrum is a nutrient-rich milk secretion containing
immunoglobulins, growth factors, and other bioactive compounds that
support calf nutrition and immune development. Studies have suggested
that bovine colostrum may also benefit human health, such as improved
immune function, reduced risk of respiratory and gastrointestinal
infections, and enhanced gut health (SANGILD et al., 2021; GHOSH \&
IACUCCI, 2021; MEHRA et al., 2022; LINEHAN, ROSS \& STANTON, 2023).

Bovine colostrum has been used as a nutritional and therapeutic
supplement in various clinical conditions in humans, such as
malnutrition, infections, inflammatory bowel disease, and
musculoskeletal injuries (PLAYFORD et al., et al., 2021). However,
further studies are necessary to fully understand the mechanisms by
which bovine colostrum exerts its beneficials effects on human health.
This study aims to review scientific articles on the use of bovine
colostrum as a bioactive agent for tissue regeneration in humans,
covering topics such as its nutritional and immunological properties,
ability to promote cellular and tissue regeneration, and potential
mechanisms involved. The goal is to provide an updated overview of the
therapeutic potential of topical use of bovine colostrum in tissue
regeneration and its clinical implications.

\textbf{METHODS}

For the evaluation of cellular and immunological components, a
systematic review was performed according to the guidelines provided in
the PRISMA-P (MOHER et al., 2015) statement to answer the following
question: ``Does the application of bovine colostrum help tissue healing
of wounds?''. To group relevant studies in the area, it was carried out
by two qualified researchers. The search was carried out in the
following databases: Web Of Science, Medline (through doing Pub Med),
Scopus, Embase and Chochrane. We took into consideration both in vivo
and in vitro studies, including articles written in English.

To conduct the research, the keywords ''bovine colostrum,'' ''wound
healing,'' and ''skin'' were used. Duplicate articles were removed, and
all relevant information was stored in an online account maintained by
the principal investigator. We evaluated all studies for eligibility
based on our acronym-based inclusion and exclusion criteria for Patient,
Intervention, Comparison, and Outcomes (PICO). For patients, we included
studies related to tissue regeneration with the search terms ''Wound
healing,'' ''skin,'' ''healing wound,'' ''healings wound,'' and ''wound
healings'' OR ''guided tissue regeneration'' and ''colostrum'' OR
''colostrums'' for intervention. For intervention, we included studies
that used some form of administration of bovine colostrum. We only
considered articles that had relevant terms related to bovine colostrum
and tissue regeneration in their title or abstract, and review articles
were excluded (Figure 1).

\textbf{RESULTS}

Twelve articles related to bovine colostrum on tissue regeneration were
included and the table below provides a comprehensive overview of the
studies found in our search that investigate the effects of colostrum in
various cell types and animal models. The table comprises details such
as author and publication year, study type (in vitro or in vivo),
utilized cell or animal model, colostrum concentration, collection time,
and each obtained result. These studies provide valuable information
about the potential benefits of colostrum, particularly when collected
in the early stages of lactation.

\textbf{Table 1 --} Selected articles in systematic review.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\textbf{Autor/ publication yerar} & \textbf{Type} & \textbf{Cellular
type or animal model} & \textbf{Colostrum concentration} & \textbf{Hour
coletted} & \textbf{Results}\tabularnewline
\midrule
\endhead
Klagsbrun 1980. & In vitro & madin-darby canine kidney epithelial cells
(MDCK), rabbit kidney epithelial cells (RK13), primary calf kidney cells
(CK), and primary bovine embryonic kidney cells (BEK). Human skin
fibroblasts. Rat embryo Fibroblasts. & 1 to 20\% & 8h after the birth of
a calf & Potentials of colostrum were observed on epithelial cell
growth, but not on fibroblast growth.\tabularnewline
Takayama, Kitsunai \& Mizumachi. 2001. & In vitro & WI-38 fibroblasts &
1\% & 4.5 hours after parturition & Colostrum presence enhanced collagen
gel contraction within the initial 12-hour period, yet no stimulation of
cell growth was noted.\tabularnewline
Kovacs et al. 2009. & In vitro & human keratinocytes (HaCaT) & 0.5 to
20\% & first hour after parturition & Incubation with 5\% and 10\%
colostrum for 24 hours triggered the proliferation and migration of
keratinocytes (HaCat cells).\tabularnewline
Doillon et al. 2011. & In vitro & human foreskins fibroblasts & 1:1000
to 1:0.5 & - & Elevated IMf concentrations (ranging from 1:10 to 1:1)
led to heightened fibroblast presence and enhanced stress fiber
formation.\tabularnewline
Kshirsagar et al. 2015 & In vivo & humam patient with deep wounds (stage
II-IV) & - & - & The application of colostrum dressing enhanced wound
healing.\tabularnewline
Tanideh et al. 2017. DOI:10.1007/s00580-016-2347-5 & in vivo & sprague
Dawley rats & 0.5--1 ml & - & The use of topical colostrum application
aided in wound reduction.\tabularnewline
Kovacs et al. 2020. & In vitro & normal human keratinocytes (NHKs) and
normal human fibroblasts (NHFs) & 0.05\% a 0.5\% & within the first
hours right after giving birth & Bovine colostrum has the capacity to
enhance keratinocyte stratification and terminal
differentiation\tabularnewline
Seo et al. 2021. & In vitro & cell line HaCaT & 1, 10, 100, 200, and 400
\selectlanguage{greek}μ\selectlanguage{english}g/mL & within 24 h after calf birth & Through activation of the JNK and
p38 MAPK pathways, the fermented colostrum whey elevated AQP-3
expression and fostered keratinocyte proliferation.\tabularnewline
\selectlanguage{ngerman}Çeleb et al. 2022. & In vivo & male wistar albino rats & - & - & The
results did not demonstrate a noteworthy reparative effect of bovine
colostrum\tabularnewline
Kim et al. 2022. & In vivo and in vitro & NIH-3T3 (murine fibroblast
cell line) SVEC4-10 (murine endothelial cell line), HS68 (human
fibroblast cell line), and PMEF (primary mouse embryonic fibroblast
cells) and BALB/c mice to in vivo study & - & early stages of lactatio &
Colostrum-derived extracellular vesicles (Colos EVs) contain a variety
of anti-inflammatory agents that assist in transitioning from the
inflammatory phase to the proliferation phase. Furthermore, these
vesicles include essential factors for tissue remodeling and
angiogenesis.\tabularnewline
Tarff et al. 2022. & In vivo & C57BL/6 mice & 5\selectlanguage{greek}μ\selectlanguage{english}l & - & Topical
application of BC facilitates corneal reepithelialization and wound
healing by suppressing the inflammatory process.\tabularnewline
Kaur et al. 2022. & In vitro and in vivo & Cell NIH3T3, wistar albino
rats & & within 10 h of delivery & Various growth factors found in
colostrum have demonstrated their capacity to stimulate skin repair and
regeneration through direct interaction with genetic
material.\tabularnewline
\bottomrule
\end{longtable}

\textbf{DISCUSSION}

\textbf{In vitro}

\textbf{Fibroblasts}

Evaluating the activity of colostrum in fibroblasts is highly valuable
since fibroblasts are essential during inflammation, cell proliferation,
and differentiation, and are responsible for deposition of extracellular
matrix (ECM) components, wound contraction, and new ECM remodeling
(GOMES et al., 2021; DESJARDINS-PARK, FOSTER \& LONGAKER, 2018). In our
search, the oldest work that used colostrum in this cell type was that
of KLAGSBRUN (1980) who used colostrum to complement the culture medium
in the growth of human fibroblasts and in four other cell lineages:
Madin-Darby canine kidney epithelial cells (MDCK), rabbit kidney
epithelial cells (RK13), primary calf kidney cells (CK) and primary
bovine embryonic kidney cells (BEK).

Contrary to expectations, sparse cultures of human fibroblasts and mouse
fibroblasts were unable to grow in DMEM supplemented with colostrum. The
ideal colostrum concentration for MDCK growth was 2.5\% (vol/vol). At
higher colostrum concentrations, adhesion of MDCK cells to the culture
flask was poor and resulted in a reduced final saturation density.
Although the colostrum prevented the growth of fibroblasts, it was not
toxic because the growth of these cells resumed when the first day's
colostrum was replaced with serum. The conclusion reached at the time
was that colostrum contains specific factors necessary for epithelial
growth, but lacks those necessary for the growth of fibroblasts
KLAGSBRUN (1980). These findings suggest that colostrum may have
promising effects on the growth of epithelial cells but might not be
suitable for the growth of fibroblasts.

Another fibroblast study was performed in 2011 with human
foreskin-derived fibroblasts, where they did not use powdered colostrum,
but a complex composite fraction (IMf) of colostrum-derived serum. This
fraction was used because, according to the authors, casein found in
colostrum may end up masking the effect of bioactive compounds, such as
insulin-like growth factor type 1 (IGF-1), and for processing the
fraction, caseins were removed (DOILLON et al., 2011). Takayama et al.
(2001) also used casein-free colostrum fractions. Other studies report
that not only caseins, but also their peptides inhibit cell
proliferation and stimulate differentiation, even if they do not show
toxic results in toxicity tests (LEDESMA-MART\selectlanguage{ngerman}ÍNEZ et al., 2019; LONETTI
et al., 2019).

Casein is a protein present in milk and its derivatives that has the
ability to inhibit cell proliferation through various mechanisms. The
protein can be broken down into peptide fractions, including
casomorphin, which has inhibitory effects on cell proliferation. In
addition, casein can bind to specific receptors on cells, such as
estrogen receptors, and inhibit their activation, decreasing the
production of cell growth factors such as epidermal growth factor (EGF).
Casein can also affect the activity of enzymes involved in cell
proliferation, such as cyclin-dependent kinases, interrupting cell cycle
progression and consequently, cell proliferation (MINEO et al., 2018;
TANG et al., 2021)

Still regarding the work carried out by Doillon et al., (2011), at the
lowest IMf dilution (1:0·5) the activity of fibroblasts in the collagen
gel was close to the control, while higher concentrations (1:10--1 :1)
induced an increase in fibroblasts and stress fiber formation that was
observed mainly in fibroblasts cultured in 5\% FBS and with high
dilutions of IMf. In general, fibroblasts had better activity at higher
concentrations of IMf, while those exposed to low dilutions of IMf
exhibited widely aggregated and elongated F-actin (DOILLON et al.,
2011).

F-actin plays a crucial role in tissue regeneration by supporting cell
adhesion, migration, and extension, which are essential processes for
successful tissue repair. During tissue repair, F-actin undergoes
dynamic changes to promote cellular movement and the formation of new
tissue. For example, in response to tissue damage, F-actin helps to form
a contractile ring that allows cells to divide and proliferate, which is
important for tissue regeneration (YADAV et al., 2019). Additionally,
F-actin helps to direct the migration of cells towards the site of
injury and provides mechanical support to maintain cell shape during
tissue remodeling (CHANG et al., 2020). Overall, F-actin is a critical
component of the cellular machinery involved in tissue regeneration and
repair.

Takayama et al., (2001) investigated the effects of bovine colostrum
serum (final concentration 1\%) on the migration of W138 human
fibroblasts and verified that when added to DMEM, it increased the
contraction of the collagen gel, with effects similar to those of TGF -ß
(10ng/ml) (control) in the first 12 hours of contraction, however, cell
growth promotion was not observed. The test carried out evaluating the
contraction of the collagen gel is a classic tool to study cells of the
extracellular matrix, where the cell-laden collagen is placed in a
container and forms a gel adhered to the bottom. From there, the
diameter of the gel surface area is used as a parameter to quantify the
level of cell contractility (ZHANG et al., 2019a).

Recently, in 2022, KIM et al., tested the ability to promote wound
repair using extracellular vesicles (EVs) from milk and bovine
colostrum, which, according to the authors, provide a cocktail of
anti-inflammatory cytokines and factors to store the remodeling of the
destroyed extracellular matrix. These vesicles are secreted by most cell
types and play an important role in cell communication. Bovine milk is
an accessible source of them, which can be obtained easily and in large
quantities by serial centrifugation and filtration steps (HAN et al,
2022; ZHANG et al., 2019b).

They verify higher concentrations of EVs were present in biological
fluids (serum or milk) compared to cell culture medium (CCM). The yield
of EVs from colostrum was about 60 times higher than that from CCM.
Then, they investigated the potential activity of colostrum EVs in the
proliferation phase of wound healing. The colostro EVs treatment caused
an approximately fourfold increase in scratch closure too (KIM et al.,
2022).

The EVs of colostrum are small liposomes that transport various
bioactive molecules, including growth factors and cytokines, which can
play an important role in tissue regeneration (MECOCCI et al., 2022).
The previous study demonstrated the ability of EVs to promote cell
proliferation, migration, and differentiation of cells involved in wound
healing and tissue regeneration (KIM et al., 2022). These properties
make colostrum EVs a potential promising therapy for the treatment of
wounds and other tissue regeneration conditions.

Based on the results of the studies found, it is evident that more
research is needed to prove the effectiveness of bovine colostrum on
fibroblasts. Although the study conducted by Klagsbrun (1980) produced
unsatisfactory results in one of the cell types, Doillon et al. (2011)
and Kim et al. (2022) obtained promising results. It is important to
note that the positive results were found in studies that used specific
portions of bovine colostrum, suggesting that although these portions
show positive results in fibroblasts, when combined with other
components of colostrum, they may be ineffective. Therefore, more
research is needed to identify and understand the specific factors
present in bovine colostrum that affect the activity of fibroblasts.

\textbf{Keratinocytes}

Keratinocytes are the most abundant cells in the epidermis and play a
critical role in tissue regeneration. During the process of tissue
regeneration, keratinocytes proliferate and migrate to the injured area
to cover the wound and restore the cutaneous barrier (WANG et al., 2021;
ZHAO et al., 2018). Additionally, these cells are capable of secreting
growth factors, such as epidermal growth factor (EGF), which stimulate
the proliferation and differentiation of other cells, including
fibroblasts and endothelial cells, as well as the production of collagen
and other components of the extracellular matrix (ECM). Recent research
has explored the use of keratinocytes in tissue regeneration, either
through autologous cell transplantation or mesenchymal stem cell therapy
(WANG et al., 2021; HEYDARI et al., 2022).

Kovacs et al. (2009) investigated the effects of bovine colostrum on
keratinocytes using HaCaT cells. Cell growth was assessed with varying
colostrum concentrations (0.5-20\%). The results showed that treatment
with 5\% and 10\% colostrum for 24 hours stimulated keratinocyte
proliferation (p\textless{}0.01), while lower concentrations (0.5\%,
1\%, and 2\%) did not have significant effects, and the highest
concentration (20\%) had an inhibitory effect after 48 hours.

With the aim of increase the results, they analyzed the growth promoting
effect induced by colostrum in immunofluorescence analysis, with the
concentrations 5\% and 10\%, during 24h. The results show that colostrum
induced increase in the number of cells. Then, they evaluated whether
colostrum affect the cell migration, and to this, they kept HaCat in
medium containing colostrum and observed the started to cover a scrat
área within 6h compared to with the presence of cells more elongated,
typical of a migratory stage and after 24h the colostrum and FBS
contributed to cell migration and also for proliferation, as expected
from the results obtained in growth promoting activity.

The same author and collaborators published a new and more comprehensive
article in 2020, using lower concentrations of bovine colostrum (KOVACS
et al., 2020). In their first study, the tested concentrations had a
wider range, from 0.5\% to 20\% (KOVACS et al., 2009), while in the
second study, the concentrations ranged from 0.05\% to 0.5\% (KOVACS et
al., 2020).

The study evaluated the effects of colostrum on human primary
keratinocytes. Colostrum concentrations of 0.3\% and 0.5\% decreased the
number of active phase keratinocytes and induced the expression of
cyclin-dependent kinase inhibitors. Colostrum induced differentiation in
keratinocytes, as demonstrated by morphological changes and increased
expression of K16, an early marker of keratinocyte differentiation.
Colostrum also increased the expression of late differentiation markers,
including involucrin and filaggrin, and facilitated the transition from
early to late stages of differentiation. These effects were observed in
a dose-dependent manner (KOVACS et al., 2020). Filaggrin is an important
protein in the formation of the skin barrier and its expression is
associated with keratinocyte differentiation and formation of the
stratum corneum, the protective layer of the skin (IRVINE, MCLEAN \&
LEUNG, 2011).

Using the 3D cell culture system with polystyrene scaffolds, the authors
were able to observe that bovine colostrum supported early
differentiation state by stimulating the expression of K1 in the
suprabasal layers, improving the terminal differentiation process by
promoting the stratification of keratinocytes and the expression of
involucrin in the outermost layers. The study also investigated the role
of calcium in the differentiation process of keratinocytes. The results
showed that the addition of bovine colostrum had more pronounced
differentiation characteristics than the addition of calcium to the
culture médium (KOVACS et al., 2020).

Bovine colostrum also increased the levels of phospho-PI3K p85(Tyr458),
phospho-Akt (Ser473), PLC-\selectlanguage{greek}γ\selectlanguage{english}1 protein, and phospho-PKC\selectlanguage{greek}α, \selectlanguage{english}indicating a
possible relationship between the signaling pathways activated by bovine
colostrum and the differentiation of keratinocytes. Additionally, the
study observed that tyrosine kinase receptors (RTKs) and G
protein-coupled receptors (GPCRs) appear to play an important role in
the initial phase of keratinocyte differentiation. Activation of the
epidermal growth factor receptor (EGFR) was also verified due to its
involvement in promoting terminal differentiation through the
PLC-\selectlanguage{greek}γ\selectlanguage{english}1/PKC\selectlanguage{greek}α \selectlanguage{english}pathway (KOVACS et al., 2020).

Other study, investigated the relationship between colostrum serum and
the expression of AQP-3 using HaCaT cells. The researchers verified the
cytotoxicity of colostrum in the cells and found that no concentration
was cytotoxic. The cells increased their viability, especially at the
highest concentration. RT-PCR was used to identify the AQP subtypes
expressed in HaCaT, where AQP-3 and AQP-10 were identified as being
expressed, with AQP-3 being the most abundant (SEO et al., 2021).

They then evaluated whether colostrum serum altered the expression of
AQP-3 at concentrations of 1, 10, or 100 \selectlanguage{greek}μ\selectlanguage{english}g/mL. Cells treated with whey
colostrum did not show a significant increase in expression compared to
the control, while cells treated with fermented colostrum serum showed
an increase in AQP-3 expression at concentrations of 10 and 100 \selectlanguage{greek}μ\selectlanguage{english}g/mL.
The duration of AQP-3 expression with fermented colostrum at a
concentration of 100 \selectlanguage{greek}μ\selectlanguage{english}g/mL was evaluated, and an increase in expression
was observed at hour 2, with a peak at hour 3 and a decrease after hour
6. Hour 3 was then selected to identify the expression of AQP-3 at the
protein level using fermented colostrum at concentrations of 0, 1, 10,
and 100 \selectlanguage{greek}μ\selectlanguage{english}g/mL (SEO et al., 2021).

The authors found an increase in AQP-3 protein expression levels after
treatment with fermented colostrum at concentrations of 10 and 100
\selectlanguage{greek}μ\selectlanguage{english}g/mL, with expression levels being 10.5 and 10.9 times higher than
those of the negative control (SEO et al., 2021).. This suggests that
fermented colostrum is positively related to AQP-3 expression in
keratinocytes. Although there is no direct evidence in this study, it is
possible that fermented colostrum regulates HaCaT keratinocyte
proliferation by positively regulating AQP-3

The EGF, which is abundantly present in colostrum, is a known upstream
regulator of AQP-3 expression in cancer cell cultures (LI et al., 2013;
SMITH et al., 2023). Additionally, lactoferrin, exopolysaccharides, and
bioactive peptides with low molecular weight are present in fermented
milk and serum, which can stimulate the proliferation and healing of
skin cells. Previous studies have reported an increase in keratinocyte
proliferation and AQP-3 expression after treatment with \textless{}3 kDa
molecular weight compounds produced during cheese fermentation
(L\selectlanguage{ngerman}ÖNNERDAL, 2010). These findings suggest that low molecular weight
peptides produced during fermentation activate keratinocytes.

The studies found in this research related to the use of bovine
colostrum in keratinotics bring not only physiological data, but there
are also works that deepened the analysis and, as a result, obtained
promising results in relation to the expression of proteins and other
growth factors that are related to the process of tissue regeneration.
In general, \emph{in vitro} studies show that bovine colostrum may have
a beneficial effect on cells, both with the use of fibroblasts and
keratinocytes. This mainly involves stimuli that decrease cell
proliferation and induce differentiation. It is worth noting that the
concentration of colostrum used has a direct impact on the results, and
extracted portions appear to yield better results than the complete
colostrum

\textbf{IN VIVO}

In our search, the in vitro studies found included studies in Sprague
Dawley rats (TANIDEH et al. 2017), Balb/c mice (KIM et al., 2022),
female guinea pigs (DOILLON et al., 2011); wistar albino rats (ÇELEBI et
al., 2022; KAUR et al., 2022), female and male healthy C57BL/6 mice
(TARFF et al., 2022) and humans (KSHIRSAGAR et al., 2015)

Kshirsagar et al. (2015) compared the use of colostrum dressing versus
conventional dressing in patients aged 20 to 60 with stage II to IV deep
wounds. The experimental group received the application of powdered
colostrum twice a day, at 7am and 7pm, for 15 days, with wound
observations on the 3rd, 6th, 9th, 12th, and 15th day. The dependent
variables were the healing process, wound size, wound impregnation,
amount and color of exudate, pain felt by the patient, and reduction in
wound stages during the days of application. The conventional dressing
was done with betadine and hydrogen peroxide. The study analyzed the
effect of powdered colostrum by comparing the dependent variables in the
experimental and control groups. The study found that the use of
colostrum dressing yielded better results, with only 20\% of patients
maintaining their lesions in weeks 3 and 4, compared to 40\% of patients
in the conventional dressing group. Between the 12th and 25th day, only
15 patients still needed colostrum dressings, as opposed to 45 patients
who needed conventional dressing. The results also showed that colostrum
dressing can reduce hospitalization time, promote ulcer healing, and
reduce pain in cases of deep ulcers. Colostrum dressing is an
economical, readily available, non-immunogenic, and easy-to-apply
option.

Kim et al., 2022 tested the Colos EVs, in wounds created in BALB/c mice
and histological analysis performed 13 days after wounding showed that
both groups treated with Colos EVs showed a thicker dermal layer and had
enhanced re-epithelialization, with more granulation tissue compared to
the group without colostrum. In this work, treatments with Colos EVs
induced elastin deposition and CD31 expression, which are indicative of
increased angiogenesis. In addition, the application of Colos EVs seemed
to accelerate wound healing in a concentration-dependent manner, where
the higher the concentration, the faster the wound healing was, even
faster than the control used with the epidermal growth factor human
recombinant (rhEGF, Nepidermin), which is commonly used as a drug to
promote skin wound healing.

The other study that used colostrum fraction was conducted by DOILLON et
al. (2011), where they treated wounds with a complex composite fraction
of colostrum and compared two different models, one treated immediately
and the other treated once closed. Results from the first model
demonstrated that treatment with IMf (1:0·5) for 23 days led to less
wound contraction when compared to treatment with PBS. However, there
was no significant difference in wound contraction when treated with
different dilutions of IMf (ranging from 1:1000 to 1:10), except for a
significant decrease in wound contraction on day 14 at 1:1 dilution of
IMf and on day 23 at the lowest dilution (1:0·5) compared to control
wounds. On day 23, histological sections showed that the areas occupied
by the wound tissues were significantly higher after treatment with 1:1
IMf compared to control wounds, but not with other conditions.

The second model showed that wounds treated with cIM after closure had a
distinct shape and scar tissue appearance compared to wounds treated
with cBSA. The surface areas occupied by residual wound areas and scar
tissues were significantly different between the cIM- and cBSA-treated
wounds. Histological sections revealed larger scar tissue in the
cIM-treated wounds, but the collagen fiber contents exhibited similar
patterns in both cIM and cBSA-treated wounds. The results obtained by
the authors are not sufficient for a complete conclusion on the use of
colostrum fraction for wound treatment, and therefore, further research
on the topic should be conducted.

The study by Çeleb et al. 2022 induced ulcerative mucositis in rats and
divided them into two treatment groups, in addition to a control group.
One group received bovine colostrum from the 1\textsuperscript{st} to
5\textsuperscript{th} day post-incision, while the other group received
it from the 5\textsuperscript{th} to 10\textsuperscript{th} day.
Histopathological and immunohistochemical analysis showed that both
treatment groups exhibited ulcers, hemorrhagic areas, necrosis, and
infiltration by mononuclear cells, as well as higher levels of TNF-\selectlanguage{greek}α \selectlanguage{english}and
8-OHdG compared to the control group. There was no statistical
difference in wound healing between the groups, but the treatment groups
had a higher incidence of severe infections compared to the control
group. The results did not indicate a significant reparative effect of
bovine colostrum on mucositis and are limited by the study. Further
studies are needed to elucidate the pathogenic inflammatory mechanisms
of mucositis and the prophylactic and therapeutic roles of antioxidants.

However, other studies have obtained good results when using colostrum
in wounds, such as the case of Kaur et al. (2022), who developed
multifunctional nanofibrous dressings conjugated with bovine colostrum
so that the bioactive factors of colostrum could act simultaneously at
the wound site in a controlled manner. The developed dressing showed
better cellular responses in terms of viability, spreading, and
migration. In vitro studies confirmed the antioxidant and antibacterial
properties of the colostrum-conjugated dressing. Moreover, rapid wound
healing, better collagen formation, and anti-inflammatory responses in
in vivo experiments confirmed better healing of rat wounds treated with
colostrum-conjugated nanofibrous dressings. The bioactive factors
present in colostrum aid the timely and orderly progression of the
healing process. Therefore, bovine colostrum-based multifunctional wound
dressings present a promising approach to overcome the challenges of
traditional wound care (KAUR et al., 2022).

Tanideh et al. (2017), used a total of 80 adult female Sprague Dawley
rats divided into four groups of 20 animals in each. without treatment
(control), treatment with honey, treatment with colostrum and treatment
with colostrum + honey. They created a circular wound in 2-cm diameter
The progressive changes in wound were monitored by a camera on days 3,
7, 14, and 21 after operation. Complete dressing was performed with
colostrum (0.5--1 ml) every day in one of the groups. The study took
place for 21 days. at the end of the study, they observed that the
topical application of colostrum helped in the wound reduction. however,
the results were even better when they tested the synergistic effect of
applying colostrum and honey together. This result opens the door to
another interesting line of research, which is the evaluation of the
effectiveness of the synergistic use of bovine colostrum with other
bioactive compounds, with the aim of enhancing its beneficial effect on
the recovery of damaged tissues.

Tarff et al. (2022) tested the action of colostrum as a topical
treatment (in drops) in the healing of acute eye wounds after burns.
Burns were created by applying 5 \selectlanguage{greek}μ\selectlanguage{english}l of 0.15 mol/L sodium hydroxide
(NaOH) drops to the conjunctiva and corneal surface for 45 s in one eye
of each animal, while the other eye was kept as a control, without burn
and receiving only saline solution. After ten days of treating mice with
5 \selectlanguage{greek}μ\selectlanguage{english}l of colostrum in the experimental group, applied four times a day,
the animals were euthanized, and eye tissues were removed from both the
experimental and control groups.

Homogenized corneas (n=6 per group) were used to quantify
pro-inflammatory and anti-inflammatory cytokines, namely tumor necrosis
factor-\selectlanguage{greek}α \selectlanguage{english}(TNF-\selectlanguage{greek}α\selectlanguage{english}), interleukin-1\selectlanguage{greek}β \selectlanguage{english}(IL-1\selectlanguage{greek}β\selectlanguage{english}), tumor growth factor-\selectlanguage{greek}β \selectlanguage{english}(TGF-\selectlanguage{greek}β\selectlanguage{english}),
and interleukin-10 (IL-10), using Mouse ELISA MAX Deluxe Sets
(BioLegend, San Diego, CA). Statistical analysis was conducted using
GraphPad Prism (Version 7.0, GraphPad Software Inc., San Diego, CA).
Results showed that the application of BC significantly improved the
fluorescence staining score for epithelial cells and conjunctival
involvement in the corneas compared to those treated with SS after burns
on days 4, 7, and 10. The BC treatment also suppressed tissue
inflammation and neovascularization, and no significant difference was
observed in corneal thickness between the burned and non-burned corneas.

Based on \emph{in vivo} studies, it can be concluded that bovine
colostrum is a promising natural source for wound healing. The bioactive
factors present in colostrum help in the timely and orderly progression
of the healing process. However, in some of the studies found in our
search, colostrum did not bring any benefit related to tissue
regeneration, although there is no explanation for this. More studies
are needed to explore the full potential of colostrum in regenerative
medicine and to develop more effective and targeted therapies for wound
healing.

\textbf{CONCLUSION}

Studies related to the use of bovine colostrum in tissue regeneration
are still scarce, and none of the studies found analyzed all stages,
from colostrum collection to its proper use, including processing and
composition. It is also possible to observe that concentrations that
induce positive results do not show a dose-dependent effect, with very
high concentrations inhibiting cell proliferation and differentiation,
as well as very low concentrations. From the results found, it is
evident that the application method also influences the final outcome,
as specific portions showed positive results in contrast to the use of
complete colostrum. This opens doors for experiments that can be
performed with different vehicles, colostrum fractions, cell types, and
concentrations, aiming to obtain better utilization of bovine colostrum
properties in tissue regeneration.

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